Reston, Va.; December 17, 2020The Society is pleased to announce and recognize the 2021 SOT Award recipients for their many accomplishments and their commitment to the field of toxicology. The 2021 awardees represent outstanding researchers in academia, industry, and government across the globe and career stages. The work of these awardees has improved human, animal, and environmental health and addresses diverse areas, such as environmental health disparities of underserved populations, toxicokinetics of xenobiotics, and reducing animal use in toxicity testing.

This yearmore than any year in the immediate pasthas illustrated the importance of scientists working to advance public health. The SOT Award recipients represent those at the forefront of basic, translational, and cutting-edge research aimed at benefiting public health, says George P. Daston, PhD, 20202021 SOT President. The SOT Awards also honor individuals who are training the next generation of scientists and the fields most promising postdoctoral and student researchers.

SOT also is proud to welcome two new Honorary members in 2021:

The 2021 SOT Award recipients and new Honorary members will be honored during the Societys Virtual 2021 Annual Meeting and ToxExpo, March 1226, 2021.

SOT AWARDS**conferred by the SOT Awards Committee

SOT Achievement Award

SOT Arnold J. Lehman Award

SOT Distinguished Toxicology Scholar Award

SOT Education Award

SOT Enhancement of Animal Welfare Award

SOT Founders Award (for Outstanding Leadership in Toxicology)

SOT Leading Edge in Basic Science Award

SOT Merit Award

SOT Public Communications Award

SOT Toxicologist Mentoring Award

SOT Translational Impact Award

SOT Undergraduate Educator Award

SUPPORTED AWARDS

Colgate-Palmolive Awards for Student Research Training in Alternative Methods

Colgate-Palmolive Grants for Alternative Research

Colgate-Palmolive Postdoctoral Fellowship Award in In Vitro Toxicology

Syngenta Fellowship Award in Human Health Applications of New Technologies

ADDITIONAL AWARDS

Toxicological Sciences Paper of the Year Award

SOT Best Postdoctoral Publication Awards

SOT Perry J. Gehring Diversity Student Travel Award

SOT Undergraduate Research Awards

More information on the 2021 Award recipients is available on the SOT website.

# # #

About SOT Awards and HonorsThe Society of Toxicology (SOT) Awards program recognizes distinguished toxicologists and students each year based on merit. In 1962, the Society inducted its first Honorary members, establishing its honors program. In 1965, the SOT Awards program was created with the establishment of two awards, the SOT Merit Award and the SOT Achievement Award, to support the furtherance of the science of toxicology. Today, the Society presents more than 20 awards that recognize achievement, facilitate travel for senior and budding scientists, and further toxicological research. Hashtag: #SOTAwards

About SOTFounded in 1961, the Society of Toxicology (SOT) is a professional and scholarly organization of more than 8,000 scientists from academic institutions, government, and industry representing the great variety of individuals who practice toxicology. SOT is committed to creating a safer and healthier world by advancing the science and increasing the impact of toxicology. The Society promotes the acquisition and utilization of knowledge in toxicology, aids in the protection of public health, and has a strong commitment to education in toxicology and to the recruitment of students and new members into the profession. SOT values diversity, equity, and inclusiveness in all their forms and promotes them as part of all Society activities. For more information about SOT, visit the Societys website or like/follow SOT on Facebook, Instagram, LinkedIn, and Twitter.

Continued here:
Scientists Advancing Public Health Research Honored with 2021 Society of Toxicology Awards - Newswise

One of the ways scientists hope to offer better treatments for vision loss is through gene therapy, where carefully selected genetic material is injected into the eyes to address mutations. Researchers have been left surprised by the effectiveness of an experimental form of this treatment, which involved an injection into one eyeball yet improved vision across both.

Gene therapies have the potential to treat all kinds of health conditions, ranging from cancer, to diabetes in dogs, to obesity and damaged spinal cords. One area where we're seeing some really exciting progress is in hereditary vision loss, with studies demonstrating the potential of gene therapy to treat color blindness, progressive retinal diseases and glaucoma, with some recently receiving approval from the FDA.

This latest study was conducted by scientists at the University of Cambridge, the University of Pittsburgh and Paris Institut de la Vision, and focuses on a form of inherited vision loss called Leber hereditary optic neuropathy (LHON). This affects around one in 30,000 people and usually occurs in young folks aged in their 20s and 30s, destroying their retinal ganglion cells and in turn the optic nerve. Once the condition takes hold, vision can deteriorate to the point where the subject is considered legally blind in just a matter of weeks, with recovery occurring in less than 20 percent of cases.

The majority of patients suffer from the same mutation affecting the MT-ND4 gene, so the researchers were hopeful of targeting this mutation as a way of improving treatment outcomes for sufferers of LHON. They trialed their gene therapy as part of a study involving 37 patients who had suffered vision loss in the preceding six to 12 months. This meant injecting a viral vector packed with a modified complementary DNA called rAAV2/2-ND4 into the vitreous cavity at the back of just one eye, with a sham treatment injected into the other eye.

We expected vision to improve in the eyes treated with the gene therapy vector only, says study author Dr Yu-Wai-Man. Rather unexpectedly, both eyes improved for 78 percent of patients in the trial following the same trajectory over two years of follow-up.

To investigate the reasons behind this unexpected outcome, the team studied the gene therapys effects in macaques, which have a similar vision system to humans. This enabled them to analyze the tissues from different parts of the eye to see how the viral vector DNA had spread. This provided evidence of interocular diffusion, with the viral vector DNA turning up in the retina, optic nerve and anterior segment of the untreated eye.

As someone who treats these young patients, I get very frustrated about the lack of effective therapies, says senior investigator Dr Sahel, from the University of Pittsburgh. These patients rapidly lose vision in the course of a few weeks to a couple of months. Our study provides a big hope for treating this blinding disease in young adults.

The research was published in the journal Science Translational Medicine.

Source: University of Cambridge

See the article here:
Single gene therapy injection surprisingly boosts vision in both eyes - New Atlas

INTRODUCTION

Centromeres have been one of the most mysterious parts of the human genome since they were characterized, in the 1970s, as large tracts of 171base pair (bp) strings called alpha-satellite monomers (1, 2). With a growing body of evidence suggesting their relevance to human diseases as sources of genomic instability or as repositories of haplotypes containing causative mutations (38), it has become more important to investigate the underlying sequence variations in centromeric regions (9, 10).

Human centromeric regions have nested repeat structures. Namely, a series of distinctively divergent alpha-satellite monomers compose a larger unit called higher-order repeat (HOR) unit, and copies of an HOR unit are tandemly arranged thousands of times to form large, homogeneous HOR arrays. While HOR units are chromosome specific and consist of 2 to 34 alpha-satellite monomers, copies of an HOR unit are almost identical (95 to 100%) within a chromosome (Fig. 1A) (1117).

(A) Schematics of a typical DNA sequence structure of human centromeric regions. The entire region consists mostly of alphoid monomers of 171 bp long. The core centromeric regions (up to several million base pairs) with an HOR structure are sandwiched by the pericentromeric (monomeric) regions, where monomers are arranged tandemly without HOR. (B) Steps for HOR encoding of long reads. Monomer-encoded reads were obtained by aligning monomer sequences into raw long reads, and then frequent patterns of assigned monomers were considered HORs. The blue pins indicate the mismatches recorded in HOR-encoded reads, which contain both single-nucleotide variations (SNVs) and sequencing errors. (C) Structures of the canonical and some variant HORs detected in chromosome X. The rectangles represent the presence of corresponding alphoid monomers. No gap is allowed between two constituent alphoid monomers to be detected as HORs. All structures are shown in supplementary figures. (D to F) Relative frequencies (per 1000 monomers) of some detected variant HORs for 36 samples in (D) chromosome X, (E) chromosome 17, and (F) chromosome 11. (G) Example of the HOR-encoded long reads containing the variant HORs. Reads from a Japanese sample (B831) contain 13m9-13 (green rectangles), a variant found in chromosome 17. They typically showed mosaicism with other variant HORs (8-, 12-, 15-, and canonical 16-mers) or purely tandem structures. Detected HORs are represented as rectangles, placed proportionally to their actual positions within reads. Reads from a Japanese, B805, show the 6-mer variant 6m1 (light blue rectangles). While the variant seemed enriched in reads, their distribution was sporadic; at most three variants were found in tandem.

The total HOR array length of each chromosome differs markedly among individuals (7, 18) and human populations (1921). Structural alterations such as unequal crossing over and/or gene conversion are thought to be among the major driving forces of this centromeric variation (22, 23). Other types of variation occur within HOR arrays, such as single-nucleotide variations (SNVs) between paralogous HOR units (21, 24, 25) and structurally variant HORs, which consist of different numbers and/or types of alpha-satellite monomers (21, 2628). However, the importance of structurally variant HORs remains unknown because they are difficult to detect comprehensively via traditional approaches such as restriction enzymes sensitive to alpha-satellite monomers, Southern blotting, or the analysis of k-mers unique to centromeric regions in short reads obtained in the 1000 Genomes Project (29).

Recently, the advent of long-read sequencing technologies has paved the way for direct, comprehensive observation of sequence variations among various human populations (3034). Long-read sequencing was capable of yielding contiguous reference sequences of centromeres for several species (35, 36), and reconstruction of whole centromeric sequences for a human haploid genome is now possible despite their idiosyncratic repeat structures (3740). While reference-quality de novo assembly of such repetitive regions remains a demanding task involving substantial manual curation (38, 41, 42), the use of unassembled long reads has promise for investigating variations within centromeric regions of diploid genomes in a cost-effective manner (43).

Therefore, we exploited a strategy of HOR encoding of unassembled long reads for comprehensive detection and quantification of variant HORs. The use of unassembled reads enabled us to analyze diploid samples without the danger of collapsing them in assemblies. In addition, the uncorrected reads could address SNVs in the HORs in an unbiased way. Here, we revealed a hidden diversity of centromeric arrays in terms of variant HORs through analysis of long reads from 36 human samples of diverse origins. We identified many previously unidentified variant HORs including some specific to a few samples, and even when variants were shared, their observed frequencies were substantially different in general.

To investigate interindividual variation within the centromeric array, we analyzed publicly available, single-molecule, real-time sequencing reads collected from 12 samples from geographically diverse origins, including three from Africa (Mende, Sierra Leone; Esan, Nigeria; and Maasai, Kenya), two from Europe (Toscani, Italy, and Finland), five from Asia (Gujarati, India; Dai, China; and three from Han, China), and two from Latin America (Puerto Rico and Peru). We also analyzed 21 newly sequenced Japanese datasets and three previously described samples: AK1 (Korea), HG002 (Ashkenazi), and CHM13 (Europe) (31, 32, 34). Thus, we analyzed a total of 36 samples (fig. S1).

First, the long reads were preprocessed in silico to filter out the noncentromeric fraction. The remaining reads were then interpreted as a series of alphoid monomers using a catalog of 58 monomers (i.e., they were represented as monomer-encoded reads) (Fig. 1B). Then, monomer-encoded reads were clustered on the basis of the composition of different monomer types. For each cluster of reads associated with one of the HOR arrays, a catalog of variant HORs was constructed by detection of frequent patterns in the monomer-encoded reads. Thus, HORs may or may not be arranged in tandems of the same type. Last, HOR-encoded reads were obtained by automatically replacing these patterns with symbols representing HORs (fig. S1).

In this analysis, we avoided chromosomes 5, 13, 14, 19, 21, and 22, in which the chromosome identity is obscured by shared HOR patterns. We mainly focused on the HOR arrays of chromosomes 11 (D11Z1), 17 (D17Z1), and X (DXZ1), which evolved from the archetypal 5-mer HOR, since the variations in these chromosomes are more divergent than those of other chromosomes associated with dimeric archetypes, whose variant HORs are more difficult to capture (16). We therefore excluded these other chromosomes to avoid drawing inaccurate conclusions.

The detected variant HORs were diverse in terms of presence and abundance among the samples. In chromosome X, the canonical HOR consists of 12 monomers; this was the most frequent pattern found in reads across all of the datasets (96.2 to 98.4% of all HOR types). In addition to the canonical 12-mer HOR, 51 variant HORs were defined, ranging in size from 2- to 23-mer (Fig. 1, C and D, and fig. S4). While some variant HORs (e.g., 10m1-4 and 17m5-1) were shared by all 36 samples, others were specific to or missing from a few samples (Fig. 1D). For example, 18m1-6 was specific to CHM13. 13m11 was found only in five samples: Esan, Maasai, Toscani, and two Japanese (B480 and B700). The 11m9 variant was shared almost universally but was absent from HG005 and B402.

For chromosome 17, 91 distinct variants were detected, ranging in size from 5- to 39-mers (Fig. 1E and fig. S5). Notably, a 13-mer variant (13m9-13; the 10th, 11th, and 12th monomers had been deleted from the canonical 16-mer) was present at high frequency in approximately half of the samples, whereas it was generally missing from other samples. Samples with the characteristic 13-mer variant exhibited a so-called haplotype II, which has an estimated allele frequency of 35% for European populations (25, 44). Prevalent variant HORs were also observed, including a 15-mer [15m(2)] and a 14-mer [14m(1)], which suggested that the canonical 16-mer was less stable than canonical HORs in chromosomes X or 11. Consequently, unlike chromosome X, the relative frequencies of canonical 16-mer HORs were highly divergent among the samples, ranging from 21.6 to 76.0%. For the remaining variant HORs, the distribution of variant HORs across the individual samples was markedly nonuniform as well (data file S1).

In chromosome 11, where the 5-mer canonical HOR (16) was the most frequent (92.6 to 99.5% of all HOR types), 23 variant HORs were detected. As with the other chromosomes investigated, variant HORs were observed at substantially variable frequency across the 36 samples (Fig. 1F and fig. S2). The most prominent difference was observed for a 6-mer variant (6m1, a duplication of the first monomer), which existed at high frequency in Toscani, Puerto Rican, Peruvian, Korean, and 11 Japanese samples; however, it was generally missing from the remaining samples. Notably, a 7-mer variant (7m1x3, the first monomer is tripled) was found only in samples with the 6m1 variant, suggesting that 7m1x3 evolved from 6m1.

To evaluate the diversity of variant HORs within a population, we quantitatively measured variation among the 21 Japanese samples. The SD of variant HOR frequency was 45.05 events per megabase (Mb), which approximated the expected density of distinct variant HORs harbored by each individual genome. We then compared our results with a recent estimate of genome-wide structural variation (SV) detection from accurate circular-consensus long reads, which obtained a reliable set of 30,000 SVs for an individual genome, with respect to a reference genome (34). The average density of SVs for each of the 23 chromosomes (autosomes and X) was 21.16 SVs/Mb (SE = 4.45 SVs/Mb); a two-tailed one-sample t test confirmed that SVs were significantly more abundant in centromeric regions than in noncentromeric regions (P = 6.51 1018). Therefore, the centromeric array appears to change rapidly in terms of variant HORs.

Together, although canonical HOR patterns were observed in all samples, noncanonical variant HORs were more dynamic overall, as they were likely to be specific to subsets of individuals across different populations or exhibited divergent frequencies even within a population, showing rapid evolution in the human centromeric arrays.

We investigated the contexts in which variant HORs were found in long reads (Fig. 1G). For example, the characteristic 13-mer variant (13m9-13) of chromosome 17 was observed in tandem or interleaved with other HORs (Fig. 1G). In contrast, the 6-mer variant (6m1) of chromosome 11 was observed only sporadically. Therefore, unlike variant 13m9-13, 6m1 appeared incapable of independent tandem expansion; it may exhibit some preference (e.g., for length) with respect to the unit of expansion. Although modes of expansion were apparently distinct depending on the type of HOR variant, we found that the same type of HOR variant was significantly enriched locally (binomial test P < 10100 for most samples with the focal variant). This finding suggests that the variant HORs had expanded locally through a series of duplication events, rather than occurring independently (data file S2).

Next, we used rare variant HORs to detect evolutionary events in human HOR arrays; these variant HORs exist at relatively low frequencies (e.g., <5 per 1000 monomers) but are shared among multiple samples. We typically observed similar HOR patterns around the same rare variant across multiple samples, which indicated that these rare variants were orthologous or paralogous (i.e., they shared the same original event that had given rise to the variant). Alternatively, these very similar patterns may have emerged independently in a recurrent manner, but this was much less plausible according to the maximum-parsimony criterion. Therefore, we compared patterns around the rare variants to understand local sequence evolution in centromeres.

As an example of the rare variants, we selected 27m12-1(2) in chromosome 17 (Fig. 1E). This variant existed in a number of contexts, although Han Chinese trio samples (HG005, HG006, and HG007) shared a homologous pattern with other variants: 14m(1), 14m10(2), and 15m(2) (Fig. 2A). The patterns, which appeared downstream from the 27-mer variant, differed slightly between HG006 (father) and HG007 (mother) by one unit of the 15-mer variant; this suggested an indel event. Of note, both patterns were observed in HG005 (son), consistent with the Mendelian inheritance of the locus.

Each variant HOR is differently colored. (A) The pattern with four SVs, 14m(1), 14m10(2), 15m(2), and 27m12-1(2), was found only in the Chinese trio (HG005 to HG007), and both maternal and paternal patterns were observed in the son. The lines between the haplotype structures indicate the position of insertion/deletion events. (B) Other distinct patterns around a rare variant, 27m121(2). A total of nine patterns are shown. Blue and red lines represent a duplication event found within the pattern observed in Toscani samples. (C) A variant HOR, 10m6+4 (light green), is found only in four Asian samples (three Japanese and a Korean). The patterns downstream of the focal SV retained homology among five loci found in the four samples.

For the same variant, 27m12-1(2), another homologous pattern was observed in eight samples (Fig. 2B). There was considerable variation downstream from the variant, which could have occurred through a series of indel events. The variation upstream appeared more complex; however, a local duplication of 20 kb was suggested within the pattern found in Toscani samples.

Furthermore, 10m6+4 in chromosome 11 was another rare variant, found only in four Asian samples (Fig. 2C). The variant shared a subsequence with the characteristic variant 6m1; it always appeared along with 6m1, suggesting that 10m6+4 had recently evolved from 6m1. We identified five loci with the variant among the four samples; the patterns downstream indicated a single indel event between loci. Two loci found in a Korean (AK1) sample seemed to be divergent from the other three Japanese loci, according to the upstream patterns.

The above examples demonstrated that we could detect evolutionary events through analysis of variant HORs and that SV was abundant within centromeric arrays. Together, we observed ongoing evolution in the human centromeric arrays, generating rare, specific, HOR patterns.

Next, we analyzed the SNV landscape among orthologous/paralogous copies of canonical HORs: 5-mers in chromosome 11, 12-mers in chromosome X, and 16-mers in chromosome 17. Here, we did not consider indels because they cannot be called confidently using long reads. Although most of the alternative bases were observed at a low frequency 3% owing to substitution errors in the long reads, we could identify prevalent SNV sites as prominent peaks in the plots (Fig. 3, A to C; figs. S6 to S9; and data file S3). Notably, those SNVs were often shared among the samples, and their frequencies were strongly correlated (Fig. 3, D to F, and figs. S10 to S13). Although SNV frequencies typically showed stronger correlations within the trio samples or within Japanese samples (fig. S14), they did not appear to reflect a geographical pattern otherwise. This finding suggests that these prevalent SNVs were present in the ancestral human population and were relatively conserved, or that a process such as gene conversion may have substantially reduced SNV diversity, in contrast to the greater structural diversity in terms of variant HORs.

(A to C) SNV landscape over the 12-mer canonical HOR in chromosome X. SNVs with a frequency of >3% are shown. The x axis is labeled with monomer index, but the actual coordinate represents position and base; for example, the alternative base G at the 20th base of the 2nd monomer is plotted at x = 3 + (20 4) + (2 800) = 1683. The y axis is the observed frequency in percentage. Four colors are used to distinguish the alternative (nonreference) bases. (D to F) Correlation of SNV frequencies. Each dot represents a single SNV (designated by a position and an alternative base). SNVs with frequencies >3% in both samples in x and y axes are shown.

Within the set of observed paralogous SNVs on canonical HORs across our dataset (36 individuals, four types of canonical HORs in chromosomes 1, 11, 17, and X), we did not observe enrichment of transitions (A/G or C/T) over transversions ([A or G]/[C or T]) or a preference of variants for CpG sites (data file S4). These rather unexpected patterns may be partly explained by the fact that these paralogous SNVs were generated not only via original spontaneous mutations but also via a series of expansion events including crossing over and gene conversion. Notably, we confirmed that the representative HOR unit sequences were already AT-rich (GC rate = 40.24 to 41.05%) and contained fewer CpG sites (fig. S15). For example, CpG was the least frequent 2-mer in all cases, at about half of the frequency of GpC. The transition of methylated CpG to TpG may have contributed to this observed pattern.

For chromosome 17, the correlation of SNV frequencies was considerably diverse, depending on the pair of samples (Fig. 4A). Samples with highly correlated SNV frequencies often shared a similar set of variant HORs (Fig. 4B). For example, 10 samples (Maasai, Esan, and 8 Japanese) were strongly correlated in terms of SNV frequencies; they also shared a characteristic pattern of variant HORs, such as the presence of the 13m9-13 variant or the absence of the 14m6-9 variant. Another 13 samples (Mende, Toscani, CHM13, Ashkenazi, Finnish, Dai Chinese, Han Chinese trio, Peruvian, and 3 Japanese) with shared SNVs exhibited the reverse pattern in terms of variant HORs. The 13m9-13 variant is a marker for a well-known alternative allele (haplotype II) for the chromosome 17 centromere in contrast to the wild-type allele (haplotype I) (25, 44). Below, we refer to haplotypes I and II as haplotypes A and B, respectively, just for a better readability. Our analysis indicated that many other variant HORs exhibited positive or negative correlations with the marker variant 13m9-13. The haplotype combination in each sample (AA, BB, or AB) was also evident in the pairwise correlation of SNV frequencies (Fig. 4, A and B). Similarly, for chromosome 11, the presence of the 6-mer variant 6m1 defined two distinct clusters of samples, which were confirmed by SV and SNV analysis (fig. S16). This clear difference between alternative haplotypes suggested that minimal or no recombination occurred between the distinct haplotypes. Thus, they act as a single genetic locus while their internal sequences undergo rapid haplotype-specific evolution.

(A) Correlation of SNV frequencies among samples on the canonical 16-mer HOR units for chromosome 17. Sample labels are colored blue (BB), black (AB), or red (AA) according to the haplotype combination inferred by SV analysis. (B) Occurrence of variant HORs in each sample serves as a fingerprint of the haplotype. SVs were clustered by co-occurrence over the samples. A-specific and B-specific variant HORs are labeled with red and blue, respectively. Blue star: The marker variant HOR for the haplotype B, 13m9-13. Darker cells indicate that they are observed with higher frequency. Sample labels are colored according to the haplotype combination (blue, BB; black, AB; red, AA). (C) Frequencies of B-specific variant HORs (in terms of generic monomers) detected in chimpanzee and humans. (D) Schematic representations of the HORs with the B-specific pattern. The numbered blocks represent the alphoid monomers (of suprachromosomal family 3), which constitute HOR patterns in humans and chimpanzees. (E) Visualization of HOR-encoded reads with the B-specific breakpoints, 9mW+(n) and 4mW+(n), n = 1,2,3,. HORs and monomers are shown according to the actual coordinates found within reads.

These haplotypes, once established, seem to follow an expected pattern. The 21 Japanese samples included 3 homozygous AA, 10 heterozygous AB, and 8 homozygous BB observed genotypes for the chromosome 17 centromere; the allele frequencies of the A and B haplotypes were 38.1 and 61.9%, respectively. According to the Hardy-Weinberg equilibrium, the expected genotype combinations for the 21 individuals are 3.05 AA, 9.90 AB, and 8.05 BB; our observed combinations exhibited almost perfect adherence to the Hardy-Weinberg equilibrium, although the sample size (n = 21) may be too small to represent a rigorous test. The allele frequency of haplotype B in the Japanese population, 26 of 42 (61.9%), was significantly higher (P = 0.000341, binomial test) than the estimated frequency for the European population (35%) (25); this might be explained by a founder effect in the Japanese population.

To determine which haplotype, A or B, was ancestral in terms of centromere sequence evolution, we performed corresponding HOR analysis using a chimpanzee (Clint) as the outgroup (45). Although chimpanzee centromeric arrays share some HOR structures with humans, we did not rely on existing information regarding HOR patterns (16). We used a set of 10 generic monomers including five monomers (W1 to W5) of suprachromosomal family 3 so that we could equally capture HOR patterns present both in chimpanzee and in humans.

Using the generic monomers, we identified HOR patterns that were shared by the human samples with haplotype B (homozygous or heterozygous) but were absent from those homozygous for haplotype A (Fig. 4C and figs. S17 and S18). These characteristic patterns shared an HOR subpattern (123411), which served as a haplotype Bspecific marker. Notably, this pattern was frequently observed in the chimpanzee (Fig. 4C and fig. S18), although the contexts in which the breakpoints occurred differed slightly in humans and the chimpanzee (Fig. 4, D and E). These findings implied that the pattern found in haplotype B was originally shared by both species, but they might have evolved into distinct HOR arrays in each species. Subsequently, haplotype A (in which the pattern was lost) had spread within the human population.

Through an analysis of centromeric arrays, we found great diversity in minor variations and widespread characteristics that are presumably of ancient origin. Collectively, these observations demonstrated the rapid, ongoing evolution of human centromeres.

The studies of variations in the centromeric arrays at the sequence level remain preliminary in a sense. For example, although we conveniently referred 5-, 16-, and 12-mer arrays as chr11, chr17, and chrX arrays, respectively, these traditional assignments may not always be true for all individual genomes. Therefore, chromosome-level reconstruction of individual genomes is crucial as well as the analysis of local variants. Because of the limited availability of sequencing data, much of our analyses relied on cell culture, where we do not know yet how stable the centromeric arrays would be. Thus, it is possible that we have overestimated the rate of change there. Ideally for understanding the biology of the centromeric arrays, it is important to use nonculture samples and to determine the presence of somatic variations precisely.

In analyzing long-read data, it is crucial to control for data errors and biases. The detection of variant HORs was less affected by sequencing errors in this study because they were characterized by a difference of at least one alphoid monomer (171 bp). In contrast, SNV quantification may have been affected by indel errors around the sites and suffered from a low signal-to-noise ratio, especially in regions with fewer variants. The recent improvement in accuracy provided by PacBio circular-consensus sequencing technology promises more faithful observation of SNVs that occur less frequently (34).

We detected variant HORs in the diploid human centromeric arrays of chromosomes 11, 17, and X using long-read data without explicit sequence assembly. We substantially increased the knowledge of variant HORs (21, 26, 27), thereby revealing unexpected diversity in human centromeric arrays through analysis of 36 individuals. Conserved homologous regions around rare variant HORs enabled us to detect ongoing structural changes among sequences in multiple samples. Similar structural changes may occur within the sea of tandem replicates of canonical HORs. Therefore, even greater hidden diversity may be present there, compared to the conservative estimates we have described. With such diversity in centromeric arrays, we hypothesize that the tandem nature of those arrays makes them extremely variable; moreover, there is sufficient information to identify individuals, similar to the use of microsatellites. Our analysis of Han Chinese trio samples and 21 Japanese samples indicated that the HOR array structure is diverse within a single population, supporting this hypothesis.

Although the centromeric arrays showed great diversity with minor SV, there were relatively conserved characteristics among samples from geographically distant populations. For example, the frequent SNVs in the most abundant HOR units were conserved across all samples; moreover, the segregation of haplotypes A and B in chromosome 17 was recapitulated in both the African samples and the Japanese population. These universal features might have spread before the relatively recent expansion of the human population out of Africa (46), unless they were acquired independently. Investigating the evolution of the segregating haplotypes more robustly would require much denser samples of human genomes including those from sub-Saharan Africa; in the present study, we focused on analyzing an available chimpanzee long-read dataset as an outgroup for the human population. Although the majority of the HOR patterns showed divergence between humans and chimpanzees, we found some common repetitive patterns. Thus, the comparison of variant HORs, not limited to canonical HORs, is useful for analysis of human and primate centromere evolution when more human and primate samples will be available.

What does it mean to have such large structural diversity in centromeric arrays? Because centromeres have a fundamental importance to proper chromosome segregation during cell division, it was once considered unusual to observe great diversity in centromeric sequences across different eukaryotic taxa (centromere paradox) (47). Centromere drive theory explained the rapid evolvability of centromeres via genetic conflict during female meiosis I, rendering the centromeres as a crux of the molecular identity of species (48). Nevertheless, growing evidence suggests that centromeres can be highly variable within a single species (5, 10, 21, 24), and our findings of diverse variant HORs add another layer of diversification. With a more comprehensive catalog of variations, we have better chances to extract new information from existing or upcoming sequencing data. If specific types of variants turn out to have functional implication, then these variants can be useful as biomarkers. Also, we expect that such markers would be helpful for tracing evolutionary events within the centromeric satellite arrays, leading to better understanding of their formation.

This great diversity suggests that centromere function may be highly robust with respect to the underlying sequence, although some variant HORs have been associated with centromere functional abnormality (25, 49). Transcription from the centromeric arrays is another intriguing phenomenon (50); we wonder whether structurally different HORs may affect transcription processes and/or functions. At the very least, we believe that a comprehensive understanding of sequence variants would improve the mapping of genomic/transcriptomic short-read data, which would ultimately benefit future studies of centromere function.

Several mechanisms can contribute to such structural diversity within centromeric sequences: unequal crossover between sister chromatids, meiotic unequal crossover, gene conversion, and homologous recombination resulting in noncrossover products, to name a few. Among them, meiotic crossovers might arguably be excluded as a major driving force because they are suppressed near centromeric regions (7, 51), and consequently, centromeric regions are reported to form large conserved linkage-disequilibrium blocks (10). On the one hand, the structural diversity within centromeric arrays can be best explained by frequent unequal crossovers between sister chromatids and gene conversions. On the other hand, centromere integrity in a human population might have been maintained through occasional gene conversions and infrequent meiotic crossovers, both of which can counteract the diversification processes by effectively homogenizing sequences among different alleles. Notably, all these mechanisms are consistent with the local, progressive expansion suggested in this study as well as in previous evolutionary analyses (52). We speculate that all these mechanisms might have contributed to the current landscape of human centromeric arrays.

Recently, a number of whole centromeric arrays reconstructed with ultralong nanopore reads and/or accurate PacBio HiFi read have been reported for a haploid genome, showing that, at last, the time is ripe to investigate centromeres in terms of sequencing technology (3740). While de novo assemblies of centromeric arrays provide unique information, it remains a nontrivial task to validate them especially for diploids. Meanwhile, the SV analysis can be a faithful representation of local features and complements the process of de novo assembly, which must be able to recover the same types and frequencies of HORs found in reads. Notably, it requires only a single SMRT Cell per sample to obtain the amount of data (10 to 40 of 3Gb human genome) used in this study. Cost-effectiveness is an important characteristic of SV analysis, making it easier to consider the scale-up.

With an increasing number of individual genomes from the same or closely related populations sequenced by long reads, one would be able to precisely observe the processes of diversification and homogenization that occur within human centromeric arrays. Therefore, such a study should provide a basis to delineate the complex mechanisms involved and to understand the true nature of centromere evolution.

In this study, we used B cells derived from Japanese people, which was distributed by the National Institute of Biomedical Innovation, Health and Nutrition, and the study was approved by The Research Ethics Committee of the Faculty of Medicine of the University of Tokyo (Human Genome/Gene Analysis Research Ethics Review; review number 19-323). For SMRTbell library preparation, B cell DNA (Japanese samples in the main text) was sheared using a Diagenodes Megaruptor 2 with software setting 75 kb and purified using a 0.6 volume ratio of AMPure beads (Pacific Biosciences, Menlo Park, CA, USA). SMRTbell libraries for sequencing were prepared using the Procedure & Checklist-Preparing >30 kb Libraries Using SMRTbell Express Template Preparation Kit protocol. Briefly, the steps included (i) DNA repair, (ii) blunt ligation with hairpin adapters with the SMRTbell Express Template Preparation Kit (Pacific Biosciences), (iii) 15-kb cutoff size selection using the BluePippin DNA Size Selection System by Sage Science, and (iv) binding to polymerase using Sequel Binding Kit 2.1, later Sequel Binding Kit 3.0 (Pacific Biosciences). SMRTbell libraries were sequenced on Sequel SMRT Cells (Pacific Biosciences) using diffusion loading, 30-kb insert size, and 600-min movies. All the other long-read data including AK1 (31), CHM13 (32), and HG002 (Ashkenazi) (34) were obtained via a public repository (Sequence Read Archive; table S1).

To enrich the centromeric reads in silico, we calculated the reference 6-mer frequency vector with the 14 typical alphoid monomers: A, B, D1, D2, J1, J2, W1 to W5, R1, R2, and M1 (table S3). We also calculated the query 6-mer frequency vector (normalized by length in base pair) and its dot product with the reference for each long read. The dot products exhibited a bimodal distribution, which represents the mixture of centromeric and noncentromeric reads. Thus, only reads with the dot product greater than a specified threshold were included in later analysis. We modified squeakr (53) to perform these steps.

To enhance the sensitivity in detection of HOR in noisy long reads, we defined chromosome-specific monomer sequences (table S2 and fig. S19). First, 10 generic monomers (the typical alphoid monomers aforementioned excluding A, B, R1, and R2) were mapped to long reads with the same parameter as described in the next subsection. Then, the reads were segregated according to chromosomes. For example, the reads from chromosome X were identified as those that contained tandems of the pattern: W1, W2, W3, W4, W5, W1, W2, W3, W4, W3, W4, and W5. Last, corresponding subsequences were extracted from the long reads, and then we took the consensus of them to obtain chromosome-specific monomer sequences. For chromosome 17, the three characteristic arrays (D17Z1, D17Z1B, and D17Z1C) were collectively analyzed because they were not distinguished from each other at our resolution. Also, noisy long reads could not clearly segregate arrays evolved from dimeric patterns by means of the generic dimeric monomers (J1 and J2 and D1 and D2). We suspect that this is because the possible combinations of those monomers were limited compared to the pentameric case (W1 to W5).

The distinct 58 monomers (table S2) were mapped by blastn (version 2.4.0+) to long reads with the following parameters:

-max_target_seqs 1000000 -word_size 7 -qcov_hsp_perc 60

Optimal assignment was calculated via dynamic programming procedure, maximizing the following quantity i(si 50) i, j, bi < bj max (0,2(ei bj)), where i indexes monomers assigned to the read, si is the BLAST (Basic Local Alignment Search Tool) score of the hit, and (bi, ei) is the region covered by the monomer. Intuitively, it tries to assign as many monomers with acceptable scores as possible, because of the first term. The second term penalized the overlaps (cf. gaps were not penalized) so that each segment of the read be assigned at most one monomer.

As related tools for analyzing centromeric repeats, there are Alpha-CENTAURI (43) and StringDecomposer (39, 54), but they serve rather different purposes; Alpha-CENTAURI detects regular and irregular HOR patterns in individual long reads, but it does not aggregate data across the reads; StringDecomposer gives us an essentially gapless decomposition of long read into a series of monomers, but it does not summarize the data as variant HORs.

The reads from chromosomes 1, 11, 17, and X were identified as those that contained >5 chromosome-specific alphoid monomers. For the analysis including the chimpanzee, the set of 10 generic monomers, D1, D2, J1, J2, W1 to W5, and M1 (16), were used instead of the chromosome-specific alphoid monomers, as the chromosome-specific monomers (derived from human samples) were not able to capture HOR structure in chimpanzee.

Then, recurrent combinations of monomers were identified as HORs. No gap of >100 bp was allowed between neighboring monomers within the detected HORs. With the list of identified HORs, reads were processed again to be encoded as series of assigned HORs plus the mismatches (SNVs) against the reference monomers. Then, these HOR-encoded reads were analyzed as described in the main text. To confirm that noisy long reads can robustly capture the characteristics of the samples, we used the HiFi data available for the CHM13 sample. The numbers of (each type of) detected variant HORs in CHM13 HiFi have higher correlations with those in CHM13 CLR (Continuous Long Read) (0.818, 0.934, and 0.860 for 12-, 16-, and 5-mer arrays, Spearman), but lower correlations with the other 35 samples that ranged from 0.306, 0.084, and 0.075 to 0.707, 0.797, and 0.701 for 12-, 16-, and 5-mer arrays, respectively. We also confirmed that the noisy long reads can detect frequent SNVs by comparing HiFi and CLR data for the CHM13 (fig. S20).

For each chromosome, we have Mi, the total number of detected monomers in individual i, and Fvi, the frequency of variant HOR v in individual i. Then, fvi=(1Mbp/171bp)Fvi/Mi is the normalized frequency v of i per 1 Mbp (million base pairs). Then, we calculated v to be the SD of fvi over the set of individuals, which served as a measure of typical variation of variant v. Last, we approximated the total variation (per 1 Mbp) for the chromosome by V = vv.

We calculated the frequency of patterns where (i) the variant is followed by the same type of variant or (ii) the variant is followed by the canonical HOR. Then, we performed binomial test against the null hypothesis where they occur randomly according to the observed frequency of HORs.

Originally posted here:
Rapid and ongoing evolution of repetitive sequence structures in human centromeres - Science Advances

4D Molecular Therapeutics raised $75 million in June to get several gene therapy programs into and through the clinic. Now, its adding a trio of executives to spearhead its work in heart, eye and lung diseases as it looks to shepherd treatments in those focus areas forward.

Robert Fishman, M.D. becomes 4Ds chief medical officer and therapeutic area head for pulmonology. He joins from Xoc Pharmaceuticals, where as chief medical officer he led phase 1 development for programs in Parkinsons disease and migraine. Before that, he headed clinical development at InterMune, overseeing the pivotal trial of Esbriet, an idiopathic pulmonary fibrosis drug now marketed by Roche.

Accelerate Biologics, Gene and Cell Therapy Product Development partnering with GenScript ProBio

GenScript ProBio is the bio-pharmaceutical CDMO segment of the worlds leading biotech company GenScript, proactively providing end-to-end service from drug discovery to commercialization with professional solutions and efficient processes to accelerate drug development for customers.

RELATED: Restoring eyesight with genetically engineered stem cells

Raphael Schiffmann, M.D., signs on as senior vice president and therapeutic area head for 4Ds cardiology stable. He was previously director of the Institute of Metabolic Disease at the Baylor Research Institute and the lead investigator of the developmental and metabolic neurology branch at the NIHs National Institute of Neurological Disorders and Stroke.

Robert Kim, M.D., joins 4D as a senior vice president and clinical therapeutic area head of ophthalmology. Hes held multiple chief medical officer roles at ViewPoint Therapeutics, Apellis Pharma and Vision Medicines, and earlier in his career worked in ophthalmology at GlaxoSmithKline, Genentech and Novartis.

The three executives arrive six months after 4D topped up its coffers with a $75 million series C round. The capital, which came two years after a $90 million B round, was earmarked to push three programs into the clinic, including two that are partnered with Roche.

Those programs include 4D-310, a treatment for Fabry disease in which patients cells accumulate a type of fat called globotriaosylceramide, and 4D-125, a treatment for the eye disease X-linked retinitis pigmentosa. Roche has the exclusive right to develop and commercialize the latter. Roche has licensed the third prospect, 4D-110, a treatment for a type of vision loss called choroideremia.

RELATED: 4D raises $90M to move gene therapies into clinical testing with AstraZeneca and Roche

The funds will also bankroll the development of 4Ds preclinical pipeline, including IND-enabling studies for 4D-710, a program in cystic fibrosis, and other candidates for neuromuscular diseases and ophthalmology.

With the addition of Robert Fishman, Raphael Schiffmann and Robert Kim to our clinical R&D leadership team, 4DMT gains not only extensive experience in clinical development and translational medicine, but also unique and specific experience within each of the initial 4DMT therapeutic areas," said 4D CEO David Kirn, M.D., in a statement.

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4D hires a trio of area heads as it ramps up its gene therapy pipeline - FierceBiotech

BOSTON and LONDON, Nov. 19, 2020 (GLOBE NEWSWIRE) -- Orchard Therapeutics (Nasdaq: ORTX), a global gene therapy leader, today announced that the U.S. Food and Drug Administration (FDA) has cleared the companys Investigational New Drug (IND) application for OTL-200, an autologous, hematopoietic stem cell, lentiviral vector-based gene therapy in development for the treatment of metachromatic leukodystrophy (MLD). The company also has applied for Regenerative Medicine Advanced Therapy (RMAT) designation for OTL-200 to help facilitate additional dialogue with the FDA on this important therapy.

MLD is a devastating and rapidly progressing disease, especially in its most severe form where it causes young children to lose skills they once had, such as the ability to walk, talk and engage with the world around them. Sadly, most of these children will pass away by the age of five, and their families are left with no real options other than palliative care, said Bobby Gaspar, M.D., Ph.D., chief executive officer, Orchard Therapeutics. We are committed to bringing OTL-200 forward as a potential treatment for children with this fatal neurodegenerative condition. The FDAs allowance of the IND associated with OTL-200 to move forward represents an important milestone on our journey, especially given our recent receipt of a positive CHMP opinion from the European Medicines Agency recommending full marketing authorization for the therapy.

As part of the IND filing, Orchard provided to the FDA data on 39 patients, including 9 patients from the U.S., who have received OTL-200 as part of clinical studies and compassionate use programs conducted at the San Raffaele-Telethon Institute for Gene Therapy in Milan, Italy. The company has post-treatment follow-up data of up to eight years in the earliest treated patients in these programs.

Based on the extensive clinical data gathered to date, we believe that OTL-200 offers tremendous potential to transform the lives of many young patients with MLD, Gaspar continued. The IND provides an opportunity for open dialogue with the FDA, allowing us to share the comprehensive data set that we have already collected in the clinical development program and to determine a path to file a Biologics License Application for regulatory approval of OTL-200 in the U.S.

About MLD and OTL-200

Metachromatic leukodystrophy (MLD) is a rare and life-threatening inherited disease of the bodys metabolic system occurring in approximately one in every 100,000 live births in the U.S. MLD is caused by a mutation in thearylsulfatase-A(ARSA) gene that results in the accumulation of sulfatides in the brain and other areas of the body, including the liver, gallbladder, kidneys, and/or spleen. Over time, the nervous system is damaged, leading to neurological problems such as motor, behavioral and cognitive regression, severe spasticity and seizures. Patients with MLD gradually lose the ability to move, talk, swallow, eat and see. Currently, there are no approved treatments for MLD. In its late infantile form, mortality at 5 years from onset is estimated at 50% and 44% at 10 years for juvenile patients.1 OTL-200 (autologous CD34+ cell enriched population that contains hematopoietic stem and progenitor cells (HSPC) transduced ex vivo using a lentiviral vector encoding the human arylsulfatase-A (ARSA) gene) is an investigational therapy being studied for the treatment of MLD in certain patients. OTL-200 was acquired from GSK inApril 2018and originated from a pioneering collaboration between GSK and the Hospital San Raffaele and Fondazione Telethon, acting through their jointSan Raffaele-Telethon Institute for Gene TherapyinMilan, initiated in 2010.

About Orchard

Orchard Therapeutics is a global gene therapy leader dedicated to transforming the lives of people affected by rare diseases through the development of innovative, potentially curative gene therapies. Our ex vivo autologous gene therapy approach harnesses the power of genetically modified blood stem cells and seeks to correct the underlying cause of disease in a single administration. In 2018, Orchard acquired GSKs rare disease gene therapy portfolio, which originated from a pioneering collaboration between GSK and theSan Raffaele Telethon Institute for Gene Therapy in Milan, Italy. Orchard now has one of the deepest and most advanced gene therapy product candidate pipelines in the industry spanning multiple therapeutic areas where the disease burden on children, families and caregivers is immense and current treatment options are limited or do not exist.

Orchard has its global headquarters in London and U.S. headquarters in Boston. For more information, please visit http://www.orchard-tx.com, and follow us on Twitter and LinkedIn.

Availability of Other Information About Orchard

Investors and others should note that Orchard communicates with its investors and the public using the company website (www.orchard-tx.com), the investor relations website (ir.orchard-tx.com), and on social media (Twitter and LinkedIn), including but not limited to investor presentations and investor fact sheets, U.S. Securities and Exchange Commission filings, press releases, public conference calls and webcasts. The information that Orchard posts on these channels and websites could be deemed to be material information. As a result, Orchard encourages investors, the media, and others interested in Orchard to review the information that is posted on these channels, including the investor relations website, on a regular basis. This list of channels may be updated from time to time on Orchards investor relations website and may include additional social media channels. The contents of Orchards website or these channels, or any other website that may be accessed from its website or these channels, shall not be deemed incorporated by reference in any filing under the Securities Act of 1933.

Forward-Looking Statements

This press release contains certain forward-looking statements about Orchards strategy, future plans and prospects, which are made pursuant to the safe harbor provisions of the Private Securities Litigation Reform Act of 1995. Such forward-looking statements may be identified by words such as anticipates, believes, expects, plans, intends, projects, and future or similar expressions that are intended to identify forward-looking statements. Forward-looking statements include express or implied statements relating to, among other things, Orchards business strategy and goals, including its plans and expectations for the regulatory approval and commercialization of OTL-200 (known as Libmeldy in the European Union (EU)) in the U.S. and EU, and the therapeutic potential of OTL-200, including the potential implications of clinical data for eligible patients. These statements are neither promises nor guarantees and are subject to a variety of risks and uncertainties, many of which are beyond Orchards control, which could cause actual results to differ materially from those contemplated in these forward-looking statements. In particular, these risks and uncertainties include, without limitation: the risk that Orchards marketing authorization application submitted for Libmeldy in the EU may not be approved by the European Commission when expected, or at all; the risk that prior results, such as signals of safety, activity or durability of effect, observed from clinical trials of OTL-200 will not continue or be repeated in Orchards ongoing or planned clinical trials of OTL-200, will be insufficient to support regulatory submissions or marketing approval in the U.S. and EU or that long-term adverse safety findings may be discovered; the inability or risk of delays in Orchards ability to commercialize OTL-200, if approved, including the risk that Orchard may not secure adequate pricing or reimbursement to support continued development or commercialization of OTL-200; and the severity of the impact of the COVID-19 pandemic on Orchards business, including on clinical development of OTL-200 and other product candidates, its supply chain and commercial programs. Given these uncertainties, the reader is advised not to place any undue reliance on such forward-looking statements.

Other risks and uncertainties faced by Orchard include those identified under the heading Risk Factors in Orchards quarterly report on Form 10-Q for the quarter ended September 30, 2020, as filed with the U.S. Securities and Exchange Commission (SEC), as well as subsequent filings and reports filed with the SEC. The forward-looking statements contained in this press release reflect Orchards views as of the date hereof, and Orchard does not assume and specifically disclaims any obligation to publicly update or revise any forward-looking statements, whether as a result of new information, future events or otherwise, except as may be required by law.

Contacts

InvestorsRenee LeckDirector, Investor Relations+1 862-242-0764Renee.Leck@orchard-tx.com

MediaChristine HarrisonVice President, Corporate Affairs+1 202-415-0137media@orchard-tx.com

1Mahmood et al. Metachromatic Leukodystrophy: A Case of Triplets with the Late Infantile Variant and a Systematic Review of the Literature.Journal of Child Neurology2010, DOI:http://doi.org/10.1177/0883073809341669

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Orchard Therapeutics Announces FDA Clearance of IND Application for OTL-200 for Metachromatic Leukodystrophy (MLD) - BioSpace

Precision medicines, such as cell therapies, remain expensive to manufacture and hard to access by patients. For example, Kymriah, the first chimeric antigen receptor (CAR) T-cell treatment approved in the United States, can have price tags as high as $475,000. Unfortunately, precision medicines are expensive to develop and manufacture, and the costs are ultimately borne by taxpayers and patients, according to The State of Personalized/Precision Medicine a report issued last year by GlobalData.

Today, companies are developing new models to lower the costs of manufacturing and bring drugs to more patients. Among them are companies developing new business models and services, innovative equipment for on-site manufacturing in hospitals, and improved formulation technology.

A key challenge for companies is scaling up the delivery of precision medicines, notes Janel Firestein, partner and life sciences industry leader at Clarkston Consulting. Companies supplying precision medicines are harvesting material for patients in a hospital or clinic, and then freezing or shipping it fresh to a contract manufacturing organization (CMO), contract development and manufacturing organization (CDMO), or other manufacturing entity.

What were seeing with a lot of our clients leveraging contract manufacturers is theyre contracting for specific slots, she says. They have x number of slots per week or month, and the scalability of that is hard.

Precision medicines are manufactured in small batches in accordance with genetic, environmental, and lifestyle factors, that is, for patients in subpopulations that meet certain well-defined criteria. (The subset of precision medicines known as personalized medicines are even more specific; that is, they are developed uniquely for each individual patient.) If a patient doesnt pass prescreening at the scheduled time, Firestein warns, the manufacturing slot for the patients treatment is lost unless the manufacturer can find another eligible patient.

Conversely, if the company is working across multiple CMOs in different countries, it needs to schedule slots in a predictable way. You need to know which slots are open, Firestein points out. You need to leverage automation and artificial intelligence to give a manufacturing view to physicians at the patient hub, so they know which dates are available and can ensure the patients cells are viable upon receipt at the manufacturing plant.

Orgenesis is among the companies turning to localization to deliver precision medicines to patients. The companys CEO and director, Vered Caplan, is a serial entrepreneur and among the top 20 inspirational leaders in advanced medicine listed in The Medicine Makers Power List 2020. Caplan has developed a point-of-care business model for hospitals that combines technological and biological development with a business strategy.

We see that centralized processing is very costly, she explains. It can be a solution for companies working in clinical trials, butonce you get to marketit is not feasible for large numbers of patients.

The companys Cell & Gene Therapy Biotech Platform incorporates the following elements: POCare Therapeutics, a pipeline of licensed cell and gene therapies (CGTs); POCare Technologies, a suite of proprietary and in-licensed technologies; and POCare Network, a collaborative, international ecosystem of research institutes and hospitals. This platform, the company asserts, is about decentralization, enabling precision medicines to be prepared on-site at hospitals.

The platform automates the production of precision medicines by validating closed box processes to reduce cleanroom footprints once the product gets to market. Caplan works to develop and commercialize drugs that can be licensed for use by hospitals in the Orgenesis network.

What we do is offer a low-cost supply platform with processing and regulatory solutions that are validated in a harmonized fashion, she details. Essentially, we take responsibility for R&D. Our hospitals are partners, and because were working in a network, the economic burden isnt high, and we can supply the therapy at a reasonable cost.

The Orgenesis approach doesnt follow the usual approach, which involves a hospital research center licensing its drug to a pharmaceutical company, which then pays the center for clinical trials. Instead, Orgenesis works in partnership with a partner hospital throughout the commercialization process. Production of the final product is automated and supplied via an on-site point-of-care processing unitreducing the complex logistics involved in transporting cells.

Fujifilm Diosynth Biotechnologies, a global CDMO, is developing a new platform to streamline the development of adeno-associated viruses (AAVs) for gene therapies. There are three methods to make AAVs, says Steve Pincus, PhD, the companys head of science and innovation. Two of the methods use viral vectors, and a third uses plasmids.

People using the latter need a source of cells and plasmids, he notes. Unfortunately, there are few licensable cell lines and few plasmid manufacturers. Consequently, as Pincus points out, If you want to manufacture your GMP plasmids at one of these, you have to wait 6 to 12 months to get in the queue.

Fujifilm wanted to tackle these problems, so it decided to license five different Rep-Cap plasmids, an adenovirus helper plasmid, and a human embryonic kidney 293 (HEK293) cell line for AAV production by plasmid transfection from Oxford Genetics. Pincus explains that by licensing these technologies, the company means to offer an HEK293 master cell bank that is well characterized and stocks GMP-grade Rep-Cap and helper plasmids, so that people can come and use those readily available reagents without having to wait 6 to 12 months, and so that the clients pay only for what they need.

To support the production of AAVs, Pincus and his team are developing specialized upstream and downstream processes. They are also developing in-process analytics for common problems in the AAV manufacturing space, such as measuring empty and full virus capsids.

Earlier this year, on September 8, Lonza announced that in a project at Sheba Medical Center in Israel, the first cancer patient received a CAR T-cell therapy that had been manufactured using the companys Cocoon platform. Cocoon is another model for distributed manufacturinga closed, automated piece of equipment for manufacturing cell therapies at the scale of a single patient, with a custom cassette that incorporates all the media, agents, and other consumables.

When you look at the way cell therapies are manufactured, one of the costs is cleanroom space, says Matthew Hewitt, PhD, head of clinical development and personalized medicine at Lonza. A cleanroom suite graded class B for air quality is noticeably more expensive than one graded class C, and the size of the room also matters. If you move to a closed or functionally closed automated platform like the Cocoon that has integrated cell culture, then you can move to cheaper cleanroom space, Hewitt asserts. or you can increase the manufacturing density in your existing cleanroom to use the space more efficiently.

Hewitt divides CAR T-cell manufacturing into a seven-step process: 1) collecting a patient sample; 2) preparing the sample for manufacturing; 3) activating the cells; 4) modifying (transducing) the cells; 5) expanding cell populations as needed for dosing; 6) washing, harvesting, and formulating the cells; and 7) dosing the patient. According to Hewitt, the steps currently automated by Cocoon include activation, transduction, and washing/harvesting/formulation. Additional automation features, he says, will debut in the coming months. Later this year, the company will begin beta testing automatic magnetic cell separation. Next year, the company plans to incorporate automated sample preparation into the Cocoons cassette.

Speaking on the future of manufacturing for precision medicine, Hewitt says he sees a role for both distributed and centralized models. Lonzas centralized facility in Houston, TX, for example, can offer standardized and well-controlled conditions, as well as an experienced team, for process development and early-stage activities.

Once you get to later stages, he points out, manufacturing needs to be moved toward the point of care to mitigate any issues with logistics. He adds that as cell therapies become more common, building enough space to process patient therapies at a centralized facility becomes increasingly impractical. Even if your centralized location served 50,000 patients a year, he says, the logistics would be a heroic endeavor.

Gene and cell therapies dont have much going on in terms of formulation, says Maria Croyle, PhD, professor of molecular pharmaceutics and drug delivery at the University of Texas at Austin. The formulation side needs to catch up.

She argues that even though precision medicines are often formulated just by adding glycerol to the cells, preparing precision medicines to dose the patient is often a complex process. When I talk about these therapies to my students, she relates, I explain that you need to thaw them out and do complicated dilutions. Its not as simple as adding 5 mL to a flask.

Precision medicines are often stored on-site in ultra-low-temperature (80C) freezers, devices that are, Croyle notes, expensive to run. The costs are often passed onto the patient. In addition, preparing the medications often involves lengthy dilution processes. Any of these medications that arent used within a couple of hours must be discarded, pushing costs yet higher.

Although some companies are moving to freeze-drying as a way to preserve living viruses and cells, preserving a live virus can take 48 to 72 hours. I had no idea until I talked to industry how much freezer dryers were a power drain, she recalls. They use a lot of electricity for 72-plus hours, and thats added to the cost of the drug.

Croyle has developed a method for stabilizing live viruses inspired by the film Jurassic Park, which depicted the recovery of dinosaur DNA from amber. She has three patents on a peelable film, inspired by amber, into which gene therapy or vaccine products can be suspended and dried within hours. You can mix them by 8 am, peel them by 3 pm, and package them to be sent off, she asserts. Its very simple and space savingits just a flat envelope with a strip of film, and it can be used in a variety of ways.

Film-packaged doses, she says, can be rehydrated to produce nasal-sprayable vaccines or injectable gene therapy solutions, or they can be placed under the tongue and upper cheek, where dissolution of the film surface releases the vaccine, activating an immune response. To commercialize the technology, she has founded Jurata Thin Film. The company is named after a mythical Lithuanian goddess who lived in an amber castle under the sea.

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Precision Medicines That Are Tailored and Off-the-Rack - Genetic Engineering & Biotechnology News

A quick read of whose wallets got thicker in the biopharma industry,from largest to smallest.

D3 Bio

Industry-veteran George Chen left a seven-year career at AstraZeneca to launch his ownShanghai-based biotech, D3 Bio, with the support ofbig-nameinvestorslikeBoyu Capital, Matrix Partners China,SequoiaCapital China, Temasek and Wuxi AppTecs Corporate Venture Fund. At this point D3 is keeping its pipeline close to the chest.But Chensays its the approach thatsunique. The company will startwith insights from clinical development and a market assessment of unmet needs and use that to guide the clinical development path. D3 will use this$200 million Series Ato build out an R&D team focused on precision medicine in the realms of immunology and oncology.

Metagenomi

Unlocking the power of microbial evolution,Metagenomimines the worlds natural microbial environment to rapidly develop effective cures to treat incurable genetic diseases. A recent$65 million Series Awill help accelerate the expansion of its gene editing systems for therapies in oncology and genetic-related diseases.This means developing a vast database of gene editing capabilities to enable unprecedented therapeutic approaches. Working with visionary investors, such as Leaps by Bayer and Humboldt Fund, will allow us to deliver on our promise to partners and fuel the development of our own pipeline of innovative curative medicines, Thomas said in a statement.

AliveCor

AI companydisguisedas a medical device companyAliveCorpicked up$65 millionto ramp up speed on their remote cardiology platform. Amidst a pandemic, telehealth appointments have been increasingly necessary.AliveCorsECG will be strengthened withcardiologicaltelehealthservices as well as with detection and condition management services. To date, its products have served more than one million customers globally, recording over 85 million ECGs.AliveCorsKardiaMobiledevice is FDA-cleared and the most clinically validated personal ECG solution in the world.

Nereid Therapeutics

Birthed fromthe work of Clifford P.Brangwynne, Ph.D., Nereid hopes to translate the therapeutic promise of biomolecular condensates from physics to physicians.The biotech will take the$50 million Series Afunding andBrangwynnesproprietary technology enabling precise measurement, interrogationand control of phase separation in cells to develop their drug discovery platform. The platform holds potential to enable completely new approaches to discovering and developing therapeutics across a wide variety of diseases, focusing first on cancers and neurodegenerative disordersaffected by phase transitions.

KiraPharmaceuticals

Backed by$46 million in financingfrom biotech entrepreneur Peter Wirth and others,Kira launchedwith a mission of pioneering a new generation of complement-targeted therapies to treat immune-mediated diseases. With the financing in hand, Kira is aiming to have three assets in the clinic within the next 18 months. The companys most advanced program, P014, is a first-in-class biologic drug with a unique mechanism of action designed to inhibit both upstream and downstream complement targets.Former Sienna Biopharmaceuticals CEO Frederick Beddingfield will be at the helm.

Adagio Medical

Adagio is singing joyfully to the tune of a $42.5 million Series Eto supportthe commercialization of itsiCLASsystem.iCLASis Adagios intelligent Continuous Lesion Ablation Systempursuing both an Investigational Device Exemption trial and a European VT CE-Mark trial."Cardiac ablation is a large and growing market that faces significant challenges including disappointing clinical outcomes, long procedure times and unsatisfactory profitability for providers," saidTuan Huynh, ofArrowMark, one of the Series E investors joining Adagios board of directors. "We believe Adagio represents a unique opportunity to transform ablation therapy and look forward to partnering with Adagio's management team to support the company's growth and commitment to addressing challenges faced by physicians and their patients."

IniPharm

Founded in 2018 with a focus on liver disease,IniPharmbrought in$35 million with a Series Afinancing roundto take its lead program through to IND filing and into clinical trials.Theprogram targets the HSD17B13 gene, which according to CEO Brian Farmer, confers pretty amazing protection against liver disease.It doesnt appear to actually prevent the diseases causation, but slows progression to more serious illness by preventing inflammation, fibrosis and cirrhosis of the liver, which are the dangerous effects of liver disease.The potential for therapies that effectively target HSD17B13 activity is significant because it is linked to a broad spectrum of liver and related diseases, said Farmer.

InterVennBiosciences

InterVennlooks to ramp up their ability to discover biomarkersand design clinical trials with the help of a little AI. Funds from a$34 million Series Bwill expand its precision medicine platformfor cancer detection.InterVennstech platform targets carbohydrates known as glycans, looking for aberrant glycosylation of certain proteins, which are implicated in a variety of disease states, including inflammation.The company'sVOCALprojectis evaluating a blood test to determine ifan ovarian tumor is benign or malignant.It is also conducting research into colorectal cancer and kidney cancer, hunting for clinically actionable biomarkers that can be used for diagnosis, prognosis, and detection of cancer recurrence, as well as predictive tests to help choose appropriate drugs.

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Biopharma Money on the Move: November 11-17 - BioSpace

CAMBRIDGE, Mass., Nov. 02, 2020 (GLOBE NEWSWIRE) -- Sarepta Therapeutics, Inc. (NASDAQ:SRPT), the leader in precision genetic medicine for rare diseases, today announced that senior management will participate in a fireside chat at the 29th Annual Credit Suisse Virtual Healthcare Conference on Monday, November 9, 2020 at 3:30 p.m. E.T.

The presentation will be webcast live under the investor relations section of Sareptas website at http://www.sarepta.com and will be archived there following the presentation for 90 days. Please connect to Sarepta's website several minutes prior to the start of the broadcast to ensure adequate time for any software download that may be necessary.

About Sarepta TherapeuticsAt Sarepta, we are leading a revolution in precision genetic medicine and every day is an opportunity to change the lives of people living with rare disease. The Company has built an impressive position in Duchenne muscular dystrophy (DMD) and in gene therapies for limb-girdle muscular dystrophies (LGMDs), mucopolysaccharidosis type IIIA, Charcot-Marie-Tooth (CMT), and other CNS-related disorders, with more than 40 programs in various stages of development. The Companys programs and research focus span several therapeutic modalities, including RNA, gene therapy and gene editing. For more information, please visit http://www.sarepta.com or follow us on Twitter, LinkedIn, Instagram and Facebook.

Internet Posting of Information

We routinely post information that may be important to investors in the 'Investors' section of our website at http://www.sarepta.com. We encourage investors and potential investors to consult our website regularly for important information about us.

Source: Sarepta Therapeutics, Inc.

Sarepta Therapeutics, Inc.Investors:Ian Estepan, 617-274-4052, iestepan@sarepta.com

Media:Tracy Sorrentino, 617-301-8566, tsorrentino@sarepta.com

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Sarepta Therapeutics to Present at the 29th Annual Credit Suisse Virtual Healthcare Conference - Yahoo Finance

SOUTH SAN FRANCISCO, Calif.--(BUSINESS WIRE)--Veracyte, Inc. (Nasdaq: VCYT), a pioneering genomic diagnostics company, announced that the Federal Joint Committee (G-BA) has approved its Prosigna Breast Cancer Gene Signature Assay. The G-BA decision to reimburse the Prosigna test will provide access to the test for all breast cancer patients in Germany with HR+/HER2- early-stage breast cancer.

The Prosigna Assay is a second-generation breast cancer test, meaning that it uses advanced genomic technology combined with clinical and pathologic features to inform next steps for patients with early-stage breast cancer. The test analyzes the activity of 50 genes known as the PAM50 gene signature, along with tumor size, lymph node involvement, and a tumor proliferation score to provide early-stage breast cancer patients and their physicians with a prognostic score indicating the probability of cancer recurrence during the next 10 years.

We are pleased with the G-BA decision, which will enable more breast cancer patients and their physicians in Germany to benefit from the genomic insights offered by our Prosigna test, said Bonnie Anderson, chairman and chief executive officer of Veracyte. Further, because Prosigna is performed by laboratories locally, this decision will enable German laboratories to deliver precision medicine solutions directly to their physician customers.

The Prosigna test is recommended in guidelines from the German Association of Gynecologic Oncology (AGO), as well as the European Society for Medical Oncology (ESMO), the American Society of Clinical Oncology (ASCO) and the National Institute for Health and Care Excellence (NICE) in the United Kingdom.

Every year around 70,000 women in Germany develop early breast cancer. In many cases, a clear therapy recommendation for or against adjuvant chemotherapy is challenging based on the clinicopathological criteria alone. The Federal Joint Committee supports the use of biomarkers, now including Prosigna, to inform treatment decisions based upon the patients individual cancer recurrence risk.

About Prosigna

Prosigna is a prognostic Breast Cancer Gene Signature assay indicated in female breast cancer patients who have undergone either mastectomy or breast-conserving therapy in conjunction with locoregional treatment consistent with standard of care, either as a prognostic indicator for distant recurrence-free survival at 10 years in post-menopausal women with Hormone Receptor- Positive (HR+), lymph node-negative, Stage I or II breast cancer or lymph node-positive (13 positive nodes, or 4 or more positive nodes), Stage II or IIIA breast cancer to be treated with adjuvant endocrine therapy alone, when used in conjunction with other clinicopathological factors.

In addition to the risk of recurrence (ROR) information, in Europe the assay provides the intrinsic subtypes of the tumor tissue within three groups low, intermediate and high. The tests performance is validated for use on the nCounter Analysis System in laboratories across Europe.

About Veracyte

Veracyte (Nasdaq: VCYT) is a global genomic diagnostics company that improves patient care by providing answers to clinical questions, informing diagnosis and treatment decisions throughout the patient journey in cancer and other diseases. The companys growing menu of genomic tests leverage advances in genomic science and technology, enabling patients to avoid risky, costly diagnostic procedures and quicken time to appropriate treatment. The companys tests in thyroid cancer, lung cancer, breast cancer and idiopathic pulmonary fibrosis are available to patients and its lymphoma subtyping test is in development. With Veracytes exclusive global license to a best-in-class diagnostics instrument platform, the company is positioned to deliver its tests to patients worldwide. For more information, please visit http://www.veracyte.com and follow the company on Twitter (@veracyte).

Cautionary Note Regarding Forward-Looking Statements

This press release contains forward-looking statements, including, but not limited to, our statements related to our plans, objectives, expectations (financial and otherwise) or intentions with respect to Veracytes Prosigna Breast Cancer Gene Signature Assay for use in predicting long-term risk of recurrence among breast cancer patients. Forward-looking statements can be identified by words such as: "anticipate," "intend," "plan," "expect," "believe," "should," "may," "will" and similar references to future periods. Actual results may differ materially from those projected or suggested in any forward-looking statements. Examples of forward-looking statements include, among others, statements regarding Veracytes belief that its Prosigna Breast Cancer Gene Signature Assay helps physicians accurately predict long-term risk of recurrence among breast cancer patients. These statements involve risks and uncertainties, which could cause actual results to differ materially from our predictions, and include, but are not limited to: Veracytes ability to achieve and maintain reimbursement coverage for its tests; the continued inclusion of its tests in recommendations of medical associations and agencies; the benefits of Veracytes tests and the applicability of clinical results to actual outcomes. Factors that may impact these forward-looking statements can be found in Item 1A Risk Factors in our Annual Report on Form 10-K filed with the SEC on February 25, 2020 and in our Quarterly Report on Form 10-Q filed with the SEC on November 2, 2020. A copy of these documents can be found at the Investors section of our website at http://www.veracyte.com. These forward-looking statements speak only as of the date hereof and Veracyte specifically disclaims any obligation to update these forward-looking statements or reasons why actual results might differ, whether as a result of new information, future events or otherwise.

Veracyte, Afirma, Percepta, Envisia, Prosigna, LymphMark, and the Veracyte logo are trademarks of Veracyte, Inc.

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Prosigna Breast Cancer Assay Now Approved for Reimbursement in Germany - Business Wire

BURLINGTON, Mass., Oct. 08, 2020 (GLOBE NEWSWIRE) -- Flexion Therapeutics, Inc.(Nasdaq:FLXN) announced today thatAdam Muzikant, Ph.D., Senior Vice President, Business Development, will present at the annual 2020 Cell & Gene Virtual Meeting on the Mesa. Dr. Muzikant will provide a review of FX201, an investigational, intra-articular, IL-1Ra gene therapy product candidate in clinical development for the treatment of osteoarthritis (OA).

The Company is conducting an open-label, Phase 1 dose-escalation trial evaluating the safety and tolerability of FX201 in patients with knee OA. The trial is intended to test low, mid and high doses of FX201 in cohorts of five to eight patients. Following the completion of the low-dose cohort and data review by an independent Drug Monitoring Committee, the trial has advanced to enrolling the mid-dose cohort. Data from the study are anticipated in 2021.

Organized by theAlliance for Regenerative Medicine, the 2020 Cell & Gene Meeting on the Mesa will be delivered in a virtual format over the course of five days beginning on October 12. The conference will feature more than 120 presentations by leading public and private companies highlighting the technical and clinical achievements in the areas of cell therapy, gene therapy, gene editing, tissue engineering and broader regenerative medicine technologies.

About FX201FX201 (humantakinogene hadenovec) is a novel, clinical-stage, intra-articular gene therapy product candidate which utilizes a helper-dependent adenovirus (HDAd) vector based on human serotype 5 (Ad5) that is designed to transfer a gene to cells in the joint to produce an anti-inflammatory protein, interleukin-1 receptor antagonist (IL-1Ra), under the control of an inflammation-sensitive promoter. Inflammation is a known cause of pain, and chronic inflammation is thought to play a major role in the progression of OA. By persistently suppressing inflammation, Flexion believes FX201 holds the potential to provide long-term pain relief and functional improvement, and to modify disease progression.

About Flexion TherapeuticsFlexion Therapeutics(Nasdaq:FLXN) is a biopharmaceutical company focused on the development and commercialization of novel, local therapies for the treatment of patients with musculoskeletal conditions, beginning with OA, the most common form of arthritis. The company's core values are focus, ingenuity, tenacity, transparency and fun. Visitflexiontherapeutics.com.

Forward-Looking Statements This release contains forward-looking statements that are based on the current expectations and beliefs of Flexion. Statements in this press release regarding matters that are not historical facts, including, but not limited to, statements relating to the future of Flexion; timing and plans with respect to the Phase 1 clinical trial of FX201; and the potential therapeutic and other benefits of FX201, are forward looking statements. These forward-looking statements are based on managements expectations and assumptions as of the date of this press release and are subject to numerous risks and uncertainties, which could cause actual results to differ materially from those expressed or implied by such statements. These risks and uncertainties include, without limitation, the fact that the impacts and expected duration of the COVID-19 pandemic are uncertain and rapidly changing; the risk that we may not be able to maintain and enforce our intellectual property, including intellectual property related to FX201; risks related to clinical trials, including potential delays, safety issues or negative results; and other risks and uncertainties described in our filings with the Securities and Exchange Commission (SEC), including under the heading Risk Factors in our Quarterly Report on Form 10-Q for the quarter ended June 30, 2020 filed with the SEC on August 5, 2020 and subsequent filings with the SEC. The forward-looking statements in this press release speak only as of the date of this press release, and we undertake no obligation to update or revise any of the statements. We caution investors not to place considerable reliance on the forward-looking statements contained in this press release.

Contacts:

Scott YoungVice President, Corporate Communications & Investor RelationsFlexion Therapeutics, Inc.T: 781-305-7194syoung@flexiontherapeutics.com

Julie DownsAssociate Director, Corporate Communications & Investor Relations Flexion Therapeutics, Inc.T: 781-305-7137jdowns@flexiontherapeutics.com

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Flexion Therapeutics to Present at the 2020 Cell & Gene Virtual Meeting on the Mesa - GlobeNewswire

MUNICH, Germany and YOKOHAMA, Japan , November 02, 2020 / B3C newswire / -- Minaris Regenerative Medicine (Minaris), a leading global contract development and manufacturing organization for cell and gene therapies, wholly owned by Showa Denko Materials Co., Ltd., announced today a total investment of 64.5 million USD to significantly expand its facilities in Europe and Asia.

European facility expansion:

A new state of the art facility will be built in the proximity of the existing site in Ottobrunn near Munich, Germany with a total investment of 40.7 million USD. The new facility will operate according to GMP standards (FDA and EMA) and be dedicated to clinical and commercial manufacturing as well as development services for cell and gene therapies. The multi-storey building with a total of 6,650 sqm will initially more than double Minaris existing capacity in Europe by providing additional clean rooms, quality control laboratories, warehousing, cryo-storage and office space. It will have a modular design with the possibilities to go from single room to ball room design and to flexibly change between grade B and grade C configuration. The new facility is expected to be operational early 2023 and will allow for additional expansion of clean rooms according to client demand and specifications, thus more than tripling the current clean room capacity.

We are very pleased to expand our capacity to support the growing demand of clients who continue to care for an increasing number of patients in the future, said Dusan Kosijer, Managing Director of Minaris Regenerative Medicine GmbH.

Asian site expansion:

A new facility will also be established adjacent to the existing facility in Yokohama, Japan allowing for an additional 4,000 sqm which will double the capacity for commercial manufacturing of regenerative medicine. The new facility is scheduled to start operations in October 2022. The investment of 23.8 million USD is part of a strategy to establish a center for cancer immunotherapy and somatic stem cells.

The European and Asian expansions complement the opening of the new commercial facility in Allendale, New Jersey, USA announced in January this year. Our investment in the facility expansions of all our three regional sites confirms our commitment to contract development and manufacturing for the cell and gene therapy industry, commented Kazuchika Furuishi, PhD, Corporate Officer and General Manager, Regenerative Medicine Business Sector of Showa Denko Materials Co., Ltd. Our global offering to our clients with sites in USA, Germany and Japan enables us to advance our clients life-saving therapeutics to patients in need around the world.

About Minaris Regenerative MedicineMinaris Regenerative Medicine is a global contract development and manufacturing organization (CDMO) for cell and gene therapies. We offer our clients high value clinical and commercial manufacturing services, development solutions, and technologies. We are pioneers in the field with more than 20 years experience providing outstanding quality and reliability. Our facilities in the US, Europe, and Asia allow us to supply patients worldwide with life-changing therapies. Minaris Regenerative Medicine is wholly owned by Showa Denko Materials Co., Ltd.

For more information, please visit http://www.rm.minaris.com

Conversion rate: 1 Euro = 1.14 UDS, 105 Yen = 1 USD

Contact

Minaris Regenerative Medicine GmbHLuc St-Onge, Ph.D.Global Head of Sales and MarketingThis email address is being protected from spambots. You need JavaScript enabled to view it.+49 (0)89 700 9608-0

Keywords: Investments; Regenerative Medicine; Genetic Therapy; Induced Pluripotent Stem Cells; Mesenchymal Stem Cells; Allogeneic Cells; Hematopoietic Stem Cells; Dendritic Cells; Adult Stem Cells; Lymphocytes; Europe; Asia; Japan; Industry

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Minaris Regenerative Medicine to Significantly Expand Manufacturing Capacity for Cell and Gene Therapies in Germany and Japan - b3c newswire

Abstract: Options for the treatment of squamous cell lung carcinoma expanded in recent years with the introduction of the immune checkpoint inhibitors into routine clinical practice in both the first- and second-line settings but are still limited. As a result, pembrolizumab, given either alone or in combination with platinum-based chemotherapy, is now a standard first-line treatment for squamous cell lung cancer. However, few options exist once patients have progressed on immune checkpoint inhibitors and chemotherapy. In this setting, the irreversible ErbB family blocker, afatinib, has a potential role as second or subsequent therapy for some patients. The Phase III LUX-Lung 8 study demonstrated that afatinib significantly prolonged progression-free and overall survival compared with erlotinib in patients with squamous cell lung carcinoma. Notably, retrospective, ad-hoc biomarker analyses of a subset of patients from LUX-Lung 8 suggested that patients with ErbB family mutations derived particular benefit from afatinib, especially those with ErbB2 (HER2) mutations. Afatinib has a manageable and predictable safety profile, and adverse events can be managed with the use of a tolerability-guided dose modification protocol. Until more data are available, afatinib could be considered as a potential second-line treatment option for patients who have progressed on combined pembrolizumab and platinum-based chemotherapy and are ineligible for more established second-line options, or as a third-line option in patients who have received first-line immunotherapy, and second-line chemotherapy or chemotherapy and antiangiogenesis therapy. However, further data are required to support the use of afatinib following immunotherapy. Given that treatment options are limited in both of these settings, investigating an agent with an entirely new mechanism of action is warranted. If available, molecular analysis to identify ErbB family mutations or the use of proteomic profiling could help to further isolate patients who are likely to derive the most benefit from afatinib.

Keywords: EGFR, NSCLC, second-line therapy, sequencing

Plain Language Summary

Patients who have just been diagnosed with the type of non-small-cell lung cancer (NSCLC) known as squamous NSCLC usually receive chemotherapy or an immune checkpoint inhibitor (for example, pembrolizumab). Immune checkpoint inhibitors may be given either alone or in combination. For patients who have stopped responding to immune checkpoint inhibitors and chemotherapy, alternative treatments are limited and needed. One possible option is afatinib, an orally administered drug that specifically targets a receptor in the cell membrane of the tumor cell, called the epidermal growth factor receptor (EGFR). In a large clinical study, patients receiving afatinib lived for longer without disease progression than did patients who received an older drug, called erlotinib that also targets EGFR. Patients treated with afatinib also lived for longer overall than did patients who received erlotinib. Evidence from this clinical study and reports of individual patients suggest that patients with certain genetic mutations that are targeted by afatinib gain particular benefit from this drug. While afatinib does cause side effects, the most common of these are generally manageable by reducing the dose and treating the symptoms of the side effects. Further research is required to support the use of afatinib after immune checkpoint inhibitor treatment. However, it is possible that afatinib may be useful for some patients who are no longer gaining any benefit from combination treatment with chemotherapy and pembrolizumab (but are not suited to the other available therapies), and for patients who have received first-line immune checkpoint inhibitors followed by chemotherapy.

Although the treatment of lung adenocarcinoma has progressed considerably in recent years, therapy for squamous cell carcinoma, the second most common type of non-small-cell lung cancer (NSCLC), lags well behind.1 As in lung adenocarcinoma, driver mutations are common in squamous cell lung cancer; however, mutations have been found in a large number of genes, including TP53, PIK3CA, CDKN2A, SOX2, CCND2, NOTCH1/2, MET, and FGFR1.24 Squamous cell lung cancer has a particularly high tumor mutational burden (TMB), even in early-stage disease, with some cohorts displaying more than 200 exon mutations per tumor.5 In addition, tumor subclones may exhibit different combinations of mutations.6 Alterations in the tumor suppressor genes, TP53 and CDKN2A, are particularly common in squamous cell lung cancer, with studies suggesting that more than half of patients with squamous cell lung cancer carry mutations in one (and potentially both) of these genes.2,4 However, as yet, no therapies targeting these mutations have been approved for squamous cell lung cancer. Less commonly, mutations are seen in the genes encoding members of the ErbB family of receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR),4 for which targeted therapy is available. However, the nature of the mutations seen in squamous cell lung cancer differs considerably from lung adenocarcinoma, where two types of EGFR mutations (L858R and deletions in exon 19) predominate.4 As a result of the highly heterogeneous nature of squamous cell lung cancer and the wide range of mutations present, this tumor is particularly challenging to treat. In this article, we review current treatment options for squamous cell lung cancer, focusing on the role of the ErbB family inhibitor, afatinib, in this therapeutic landscape.

During the development of this review, we searched the published literature (English language only) for articles and presentations that reported clinical efficacy and safety of the second-generation EGFR tyrosine kinase inhibitor (TKI) afatinib in patients with advanced squamous cell carcinoma of the lung. Relevant publications were identified by searching the US National Library of Medicine (NLM) PubMed database, using combinations of the search terms [afatinib] AND [NSCLC] OR [squamous lung]. Reports of clinical trials and real-world evidence (case studies) were included. Other relevant publications were identified from citations in the key publications identified via NLM PubMed and from expert guidelines. Further information was obtained from the US prescribing information for afatinib.7

For patients testing positive for sensitizing EGFR mutations, anaplastic lymphoma kinase (ALK) gene rearrangements, ROS proto-oncogene 1 (ROS1) gene rearrangements, B-RAF proto-oncogene, serine/threonine kinase mutations (BRAFV600E), or neurotrophic receptor tyrosine kinase (NTRK) gene fusions, therapy options are targeted to the specific genetic aberration, as follows: gefitinib, erlotinib, icotinib, afatinib, dacomitinib, or osimertinib for EGFR mutation-positive patients; crizotinib, ceritinib, alectinib, brigatinib, or lorlatinib for patients with ALK rearrangements; crizotinib, ceritinib, or entrectinib for patients with ROS1 rearrangements; dabrafenib in combination with trametinib for patients with BRAFV600E mutation; and larotrectinib or entrectinib for patients with NTRK gene fusions. However, as targetable genetic aberrations are not identified in most patients with advanced squamous cell lung cancer,4,8,9 systemic chemotherapy and more recently, immunotherapy, are the mainstay of treatment.

First-line therapy in patients without targetable mutations is generally determined by the level of programmed death ligand-1 (PD-L1) detected by immunohistochemical staining of tumor tissue. The use of immunotherapy in the first-line setting is supported by large Phase III studies demonstrating notably extended survival with regimens incorporating immune checkpoint inhibitors (Table 1). Of note, pembrolizumab is used in combination with carboplatin and either paclitaxel or nab-paclitaxel as first-line treatment for patients with metastatic squamous NSCLC, irrespective of PD-L1 level.10 In addition, pembrolizumab monotherapy may be used as first-line treatment in patients with PD-L1 tumor proportion score (TPS) 1%,10,11 although monotherapy is generally preferred only when PD-L1 TPS is 50%.12 Recently, the FDA approved two additional first-line therapies: nivolumab plus ipilimumab (PD-L1 1%)1315 and atezolizumab monotherapy in patients with high PD-L1 expression (PD-L1 stained 50% of tumor cells [TC 50%] or PD-L1 stained tumor-infiltrating immune cells [IC] covering 10% of the tumor area [IC 10%])16,17 (Table 1). For patients with contraindications to immunotherapy, such as autoimmune disease or previous solid organ transplant, combination cytotoxic chemotherapy is recommended.18

Options for second and subsequent treatment lines depend on the first-line therapy; agents with a different mode of action are generally recommended. For patients treated with chemotherapy in the first-line, options include nivolumab or atezolizumab for any level of PD-L1 expression,19,20 pembrolizumab if PD-L1 TPS is 1%,21 and the EGFR TKI, afatinib.7,12 For patients who received immunotherapy in the first line, docetaxel combined with ramucirumab has become an established second-line option.12,22-24 Further options include docetaxel or gemcitabine monotherapy, platinum-based chemotherapy (if not already received in combination with immunotherapy in the first line), and the ErbB family inhibitor, afatinib may also be considered suitable for further investigation in this setting.7,12

The human EGFR family is composed of four members that belong to the ErbB protein lineage: EGFR (ErbB1/human epidermal growth factor receptor [HER]1), ErbB2 (HER2/NEU), ErbB3 (HER3) and ErbB4 (HER4).25 These receptor tyrosine kinases bind several growth factors, including EGF and transforming growth factor beta, forming a range of homo- and heterodimers that trigger downstream signaling pathways involved in cellular growth and proliferation. These pathways include the phosphatidylinositol 3-kinase/Akt (PKB) pathway, the Ras/Raf/MEK/ERK1/2 pathway, and the phospholipase C (PLC) pathway.

Increased expression or mutations in the ErbB family of receptor tyrosine kinases have been implicated in numerous malignancies, including lung, breast, stomach, colorectal, and pancreatic cancers, resulting in the development of a number of agents specifically targeting these receptors or their ligands (Figure 1).25 Although EGFR mutations are relatively rare,4 studies suggest that EGFR is often overexpressed in squamous cell lung cancer.26 In addition, EGFR gene copy number appears to be elevated in up to a quarter of patients with squamous cell lung cancer,4,27 and has been shown to correlate with EGFR expression.26 Studies have shown that, in addition to EGFR, other members of the ErbB family (such as ErbB2 and ErbB3) may be over-expressed or mutated in around 20% of patients with squamous cell lung cancer.2832 As a result, agents targeting EGFR have been investigated for possible use in squamous cell lung cancer (Table 2). The SQUIRE study in particular, suggested that EGFR was a valid therapeutic target in squamous cell lung cancer, with statistically significant increases in survival seen with first-line necitumumab plus platinum-based chemotherapy versus chemotherapy alone.33 However, in the FLEX and BMS099 studies, which compared treatment outcomes with cetuximab monotherapy or cetuximab combined with platinum-based chemotherapy in patients with NSCLC, subset analyses of patients with squamous cell lung cancer indicated no significant difference in overall survival (OS) between the two treatment groups.34,35 Biomarker analyses from studies of anti-EGFR monoclonal antibodies suggested that patients with elevated EGFR expression or gene copy number derived greater benefit from anti-EGFR treatment than those with low or no EGFR expression or EGFR amplification,3638 with results from the SQUIRE study suggesting little or no benefit for patients not expressing EGFR.39

Based on results from a number of studies in NSCLC that included patients with squamous cell lung cancer,4043 small molecule EGFR TKIs are not recommended for use as monotherapy or in combination with chemotherapy in the first-line treatment of unselected patients with squamous cell lung cancer. However, data from sub-analyses of studies investigating the second- or third-line use of EGFR TKI monotherapy in patients with NSCLC suggest a potential role for these agents in pre-treated patients. Significantly longer survival was seen in ever-smokers with squamous histology who received the reversible, first-generation EGFR-specific TKI, erlotinib, versus placebo, and a reduced risk of progression was observed in squamous cell lung cancer patients overall.44,45

In the Phase III TAILOR study, erlotinib was compared with docetaxel as second-line treatment of patients with wild-type EGFR and advanced NSCLC.46 Among the overall study population, erlotinib was shown to be inferior to docetaxel, producing significantly shorter OS and progression-free survival (PFS). However, in the subset of patients with squamous cell lung carcinoma, OS was similar in the erlotinib and docetaxel groups (hazard ratio [HR]=0.90 [95% confidence interval {CI}=0.491.65]), suggesting that the differences in PFS and OS seen in the overall population were driven by inferior outcomes in the erlotinib arm among patients with adenocarcinoma (~69% of the study population). Although overall survival was similar between the two treatment arms in the squamous cell carcinoma patients, erlotinib appeared to be better tolerated than docetaxel across the entire population.

Another study (PROSE) comparing erlotinib and docetaxel for the second-line treatment of unselected patients with NSCLC used the commercially-available VeriStrat serum protein test to classify patients according to whether they were likely to have a good or poor outcome after treatment with EGFR TKIs.47 VeriStrat uses matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry to measure acute-phase reactant proteins in the blood and assign a Good (VS-G) or Poor (VS-P) classification.48 PROSE was a prospective, randomized, multicenter, Phase III study that stratified patients according to a minimization algorithm by Eastern Cooperative Oncology Group (ECOG) performance status, smoking history, center, and masked pretreatment serum protein test classification.47 The proteomic test classification was masked for patients, and investigators who gave treatments, and treatment allocation was masked for investigators who generated the proteomic classification. This study showed no differences in OS between treatment groups in patients classified as VS-G (adjusted HR=1.06 [95% CI=0.771.46], P=0.714). However, OS was longer with docetaxel than erlotinib in patients classified as VS-P (HR=1.72 [95% CI=1.082.74], P=0.022), indicating that chemotherapy is a better choice in these patients.47 A more recent randomized, Phase III study, conducted in patients with advanced squamous cell lung carcinoma supported these findings, with comparable PFS and OS with erlotinib and docetaxel seen in VS-G patients.49 In this study, however, no difference in survival between the treatment arms was seen in patients classified as VS-P. Across the entire study population and within each treatment arm, survival was significantly longer in VS-G patients compared with VS-P patients (median OS, 8.2 versus 5.2 months).

Afatinib is a second-generation, irreversible ErbB family blocker that inhibits signaling from all ErbB hetero- and homodimers,50 conferring a wider inhibitory profile than first-generation, reversible EGFR-specific agents such as erlotinib and gefitinib.51 Afatinib has shown considerable efficacy in patients with EGFR mutation-positive NSCLC, and is approved as first-line treatment in this indication.7 In patients with NSCLC and sensitizing mutations in the EGFR gene, afatinib has been shown to significantly prolong median PFS compared with platinum-based chemotherapy,52,53 and a significant OS improvement has been observed with afatinib in patients with tumors harboring the exon 19 deletion (Del19) EGFR mutation.54 Further, the randomized Phase IIb LUX-Lung 7 trial demonstrated that afatinib was associated with significantly longer PFS than gefitinib.55 Afatinib is also the only EGFR TKI with United States Food and Drug Administration (US FDA) approval for uncommon EGFR mutations based on PFS and response rate.7

Although afatinib is not recommended as first-line therapy for unselected patients with squamous cell lung cancer and wild-type EGFR,12,18 it has demonstrated efficacy as second-line therapy in patients with metastatic squamous cell lung cancer following progression on platinum-based chemotherapy, and is approved by the US FDA for use as monotherapy in this patient population.7 However, despite the US FDA approval status, the inclusion of afatinib as a second-line treatment option for patients with squamous cell lung cancer varies across treatment guidelines, reflective of the changing treatment landscape in recent years. For example, afatinib is no longer included as a second-line treatment option for patients with metastatic squamous cell non-small-cell lung cancer in the NCCN Clinical Practice Guidelines In Oncology (NCCN Guidelines) Version 6.2020.56 Conversely, the latest ESMO Clinical Practice guidelines (September 2019) state that afatinib could be a therapeutic option for patients with advanced squamous cell lung cancer with unknown or wild-type EGFR status progressing on/after chemotherapy, who are unfit for further chemotherapy or immunotherapy.57

LUX-Lung 8

The approval of afatinib for use in patients who have progressed on platinum-based chemotherapy was based on results from the open-label, Phase III LUX-Lung 8 study, which compared the second-line use of afatinib (n=398) with erlotinib (n=397) in patients with advanced squamous cell lung cancer.58 Median PFS was longer with afatinib compared with erlotinib (2.4 months [95% CI=1.92.9] versus 1.9 months [95% CI=1.92.2]; HR=0.82 [95% CI=0.681.00], P=0.0427), as was OS (median 7.9 months [95% CI=7.28.7] versus 6.8 months [95% CI=5.97.8]; HR=0.81 [95% CI=0.690.95], P=0.0077; Figure 2). Although the proportion of patients with an objective response did not differ significantly between the treatment groups (6% versus 3%, P=0.055), the disease control rate was significantly higher in the afatinib group (51% versus 40%, P=0.002).

Overall adverse event profiles were similar between the two treatment arms, with 57% of patients in each group experiencing a grade 3 adverse event. However, afatinib was associated with higher incidence of grade 3 treatment-related diarrhea (10% versus 3%) and grade 3 stomatitis (4% versus 0%) than erlotinib (Table 3). Overall, 27% of afatinib-treated patients and 14% of erlotinib-treated patients underwent dose reduction due to adverse events, and 20% and 17% of patients, respectively, discontinued treatment because of adverse events.

Data on patient-reported outcomes from LUX-Lung 8 suggest that the higher rate of adverse events with afatinib did not impact on symptom scores or quality of life, as assessed by the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30 and its lung cancer-specific module, the QLQ-LC13.59 Moreover, significantly higher proportions of patients in the afatinib group than in the erlotinib group reported improved scores on the global health status/quality of life (36% versus 28%, P=0.041), cough (43% versus 35%, P=0.029), and dyspnea walked scales (35% versus 27%, P=0.022); differences in the frequency of improvements in other scales, including pain (40% versus 39%) and dyspnea (51% versus 44%), were not significant. Time to deterioration of dyspnea was significantly longer in afatinib-treated patients (median 2.6 versus 1.9 months, P=0.008).

Initial biomarker analyses using archival tissue from a subset of patients in LUX-Lung 8 indicated that the observed responses to afatinib were unlikely to be related to EGFR mutation or amplification.58 Additional analysis, conducted by Foundation Medicine (Cambridge, MA, USA) using next-generation sequencing, of a separate cohort of patients from LUX-Lung 8 that was enriched for patients with PFS >2 months indicated that these patients harbored a range of mutations, including TP53 (87% of patients), LRP1B (39%), KMT2D (33%), CDKN2A (29%) and FAT3 (26%).32 Among the 245 patients undergoing molecular analysis, 22% had tumors with at least one ErbB family mutation, including a small proportion with mutations in more than one ErbB gene, and 7% of patients had at least one EGFR mutation. In the afatinib arm, both PFS (median 4.9 versus 3.0 months, P=0.06) and OS (median 10.6 versus 8.1 months, P=0.21) were numerically longer in patients who had ErbB mutation-positive tumors (n=25) compared to those without ErbB mutations (n=107). In contrast, PFS and OS were similar in patients with (n=28) and without (n=85) ErbB mutations in the erlotinib arm (median PFS: 2.7 versus 2.5 months, P=0.29; median OS: 7.2 versus 6.4 months, P=0.46). Interestingly, the enhanced benefit of afatinib over erlotinib in patients with ErbB mutation-positive tumors appeared to be driven by mutations in HER3, HER4, and, in particular, HER2, rather than EGFR. Among 12 patients with HER2-positive tumors, PFS (HR=0.06 [95% CI=0.010.59], P=0.02) and OS (HR=0.06 [95% CI=0.010.57], P=0.02) significantly favored treatment with afatinib over erlotinib. In contrast, EGFR overexpression did not predict PFS or OS benefit with afatinib over erlotinib.

Another retrospective analysis of LUX-Lung 8 was conducted using the VeriStrat serum protein test.48 Among 412 (afatinib, n=207; erlotinib, n=205) patients classified as VS-G, OS was significantly longer with afatinib versus erlotinib (median 11.5 versus 8.9 months; HR=0.79 [95% CI=0.630.98], P not reported]). In the VS-P group (afatinib, n=129; erlotinib, n=134), there was no significant difference in OS between afatinib and erlotinib (median 4.7 versus 4.8 months; HR=0.90 [95% CI=0.701.16], P not reported). Multivariate analysis showed that VeriStrat classification was an independent predictor of OS in afatinib-treated patients, regardless of ECOG performance status or best response to first-line therapy. Together, these findings suggest that certain groups of patients with squamous cell lung cancer, such as those with HER2 mutations and those classified as VS-G, may derive particular benefit from afatinib.

It is important to note that the LUX-Lung 8 study was performed when the first-line standard of care for unselected patients with squamous cell lung cancer was chemotherapy. The treatment landscape has markedly expanded since LUX-Lung 8 was conducted; most notably, immune checkpoint inhibitors with or without chemotherapy are now available as first- and second-line treatment options, and erlotinib would no longer be considered a relevant comparator for second-line treatment in a prospective clinical trial. Docetaxel in combination with ramucirumab is now an established second-line treatment; however, at present there are no prospective, clinical data comparing afatinib with docetaxel alone or in combination with ramucirumab.

Safety of Afatinib and Use of the Tolerability-Guided Dose Modification Protocol

Afatinib has an established, predictable, and manageable safety profile that is consistent with its mode of action.52,53 No new safety signals were observed in patients with squamous cell lung cancer in LUX-Lung 8, with diarrhea (all grades/grade 3: 70/10%), rash/acne (67/6%), and stomatitis (29/4%) being the most common adverse events with afatinib (Table 3).58

Although afatinib can be associated with some severe treatment-related adverse events, following the established tolerability-guided dose modification protocol can help mitigate these reactions and allow patients to remain on treatment for as long as possible.53 According to this protocol,7 afatinib should be withheld for: any adverse reactions of grade 3; diarrhea of grade 2 persisting for 2 consecutive days while taking anti-diarrheal medication; cutaneous reactions of grade 2 that last >7 days or are intolerable. Treatment should be resumed at a reduced dose when the adverse reaction has fully resolved, improved to grade 1, or returned to baseline. Dosing should be reduced by 10 mg decrements, to a minimum of 20 mg/day. Results from several studies in patients with EGFR mutation-positive NSCLC have shown that dose reductions reduce the incidence and severity of treatment-related adverse events, without reducing the efficacy of afatinib.6062

Although dose reductions in LUX-Lung 8 occurred more frequently in patients treated with afatinib (27%) than with erlotinib (14%),58 this may have been due to the availability of multiple dose formulations of afatinib and the clear dose modification guidelines in the accompanying prescribing information.7 The implementation of these guidelines may underlie the finding that similar proportions of patients in the afatinib and erlotinib groups discontinued treatment due to adverse events (20% versus 17%), despite the fact that more patients in the afatinib group than the erlotinib group experienced grade 3 treatment-related adverse events (27% versus 17%) and/or required dose reductions.58

As noted previously, because the LUX-Lung 8 study was conducted before immunotherapy became the mainstay for the first-line treatment of advanced squamous cell lung cancer, there are no clinical trial data investigating the effect of prior immunotherapy on safety outcomes with afatinib.

Evidence from Individual Patient Cases

No additional clinical trial data on the use of afatinib as second-line treatment of advanced squamous cell lung cancer are available. As such, reports from the real-world clinical setting provide important information on treatment outcomes with second-line afatinib following chemotherapy or immunotherapy. In these settings, a number of patient case examples support the use of afatinib in patients with particular clinical characteristics, including ErbB family mutations. For example, afatinib given after chemotherapy, antiangiogenesis therapy, and icotinib successfully stabilized EGFR and HER2 mutation-positive squamous cell lung cancer in an elderly Chinese patient for at least 8 months, with no treatment-related adverse events.63 Further details have also been published of a patient enrolled in LUX-Lung 8, with multiple genetic aberrations, including EGFR copy number amplification and mutations in ErbB4, ALK, RET and BRCA. This patient experienced prolonged PFS (14.7 months) and OS (17.7 months) with afatinib;64 of note, final analysis of LUX-Lung 8 has since identified 21 patients who remained on afatinib treatment for at least 12 months.65

Afatinib has also provided clinical benefit to patients without detectable genetic anomalies, including a patient who had received chemotherapy, radiotherapy, and radiosurgery, and subsequently developed hemoptysis following treatment with nivolumab.66 This patient, who had no detectable EGFR or ALK aberrations, experienced symptomatic relief from dysphonia shortly after commencing afatinib, with no obvious adverse effects. Afatinib was given to another elderly patient who had experienced disease progression and left lung atelectasis following first-line nab-paclitaxel, resulting in resolution of the atelectasis and shrinkage of the central tumor mass, with no adverse effects.67

Personalized treatment based on validated predictive biomarkers as well as individual characteristics is nowadays the optimal approach for the treatment of NSCLC. Unfortunately, unlike for patients with adenocarcinoma NSCLC, to date, no predictive genomic biomarkers have been identified for NSCLC of squamous cell histology. Hence, cytotoxic chemotherapy and immune checkpoint inhibitors are the established gold standard for the first-line treatment of most patients with advanced squamous cell lung cancer,12,18 with the choice of regimen dependent on many factors, including the patients age, performance status, and PD-L1 TPS. Following progression on first-line therapy, molecular and physical characteristics may preclude use of further chemotherapy, and alternative treatments will be required for some patients. Alternative options will also be required to treat patients for whom immunotherapy is contraindicated, such as those with autoimmune disease.

For certain patients who are not candidates for cytotoxic chemotherapy or immunotherapy and have a good performance status, afatinib may represent a convenient second- or third-line treatment option. The challenge for clinicians is identifying these patients in routine clinical practice, and further research into predictive biomarkers that can be easily applied in the clinic is clearly needed. The Veristrat proteomic test has been validated and is covered by payors in the USA, including Medicare and Medicaid; the turnaround time is approximately 72 hours. As discussed above, having a patient with VS-G classification will give a level of comfort to physicians to treat the patient with an EGFR TKI over systemic chemotherapy. Moreover, evidence from patient case studies suggests that some unselected patients have experienced long-term benefit from afatinib, with minimal toxicity, suggesting that a trial may be worthwhile in patients who are not candidates for other therapies.

Until more data are available, afatinib could be considered a potential second- or third-line treatment option for some patients who are not eligible for other more established therapies. For example, as a second-line option for patients who have progressed on combined chemo-immunotherapy and who are ineligible for docetaxel plus ramucirumab, and as a third-line option in patients who have received first-line immunotherapy and second-line chemotherapy (e.g., docetaxel, gemcitabine or platinum-based chemotherapy) or chemotherapy and antiangiogenesis therapy (e.g. docetaxel plus ramucirumab). Due to the currently limited range of second- and third-line treatment options, investigating an agent with an entirely new mechanism of action is warranted, particularly in patients with physical or molecular characteristics that preclude the use of chemotherapy. Also, if available, molecular analysis to identify ErbB family mutations could help to further identify patients who may be likely to derive the most benefit from afatinib, in addition to Veristrat profiling as previously discussed. Importantly however, further data are required to establish the optimal place for afatinib in the squamous cell lung cancer treatment landscape, specifically among the first- and second-line treatment options that have emerged in recent years.

Afatinib may also be of value for patients who find that intravenous administration of chemotherapy and immunotherapy is logistically problematic (for example, if there is a preference or need to restrict travel to the clinic for drug infusion), or substantially impacts on their quality of life. Studies suggest that oral therapies are generally preferred by patients,68,69 and may improve quality of life since oral drug administration is more convenient and flexible.68,70 Further, oral treatment eliminates the risks and discomfort associated with intravenous administration, such as phlebitis, pain, infection, bleeding, infusion reactions, and vascular damage, and frees up valuable healthcare resources.11,69-71

No cost-effectiveness data on the use of afatinib as second-line treatment of advanced squamous cell lung cancer in the US are currently available, and further data are required in this respect. However, analyses of the LUX-Lung 8 study, undertaken from the perspective of patients treated in France and China, suggest that afatinib may be cost-effective in those countries.72,73 The French analysis calculated a 97% probability of afatinib being cost-effective, assuming a willingness-to-pay threshold of EUR70,000 per quality-adjusted life year gained.72

A number of trials are ongoing or recently completed that may offer further options for patients with squamous cell lung cancer. Results from the Phase III CHECKMATE-227 study enrolling chemotherapy-nave patients with stage IV NSCLC have led to nivolumab plus the anti-cytotoxic T-lymphocyte-antigen (CTLA) 4 monoclonal antibody, ipilimumab, being recently approved by the FDA as a first-line treatment option for patients with PD-L1 1%. In the most recent analysis, nivolumab plus ipilimumab was shown to prolong median OS relative to platinum-based chemotherapy in patients with PD-L1 expression 1% (17.1 versus 14.9 months, P=0.007) and in patients with PD-L1 <1% (17.2 versus 12.2 months, P not reported).1315 Nivolumab in combination with chemotherapy, however, did not prolong survival relative to chemotherapy alone.74

In the second-line setting in patients with squamous cell lung carcinoma, the ipilimumab plus nivolumab combination does not appear to offer any advantages over nivolumab alone. Results from a non-biomarker-matched substudy of the Phase III Lung-MAP umbrella trial showed that adding ipilimumab to nivolumab in previously treated but immunotherapy-nave patients with advanced squamous cell lung carcinoma with any PD-L1 level did not enhance survival.75 Further findings from the biomarker-driven Lung-MAP study, which is currently investigating a number of different targeted therapies in NSCLC, including durvalumab plus tremelimumab and rucaparib, may further advance the use of personalized therapy in squamous cell lung carcinoma.12,76

Results from the Phase III IMpower110 study, enrolling chemotherapy-nave patients with stage IV NSCLC, has led to recent FDA approval of atezolizumab monotherapy as a first-line treatment option for patients with high PD-L1 expression. Atezolizumab monotherapy was shown to significantly prolong median OS relative to platinum-based chemotherapy in patients with high PD-L1 expression (20.2 versus 13.1 months, P=0.0106). Primary analysis of the Phase III IMpower131 study suggested that the addition of atezolizumab to platinum-based chemotherapy in the first-line treatment of advanced squamous cell lung cancer prolonged survival.77 Median PFS with atezolizumab plus chemotherapy was 6.3 months compared with 5.6 months in patients receiving chemotherapy alone (HR=0.71 [95% CI=0.600.85], P=0.0001).77 However, final OS analysis suggested that the addition of atezolizumab only prolongs OS in patients with high PD-L1 levels, with median OS of 14.2 months in patients receiving chemotherapy plus atezolizumab compared with 13.5 months (HR=0.88 [95% CI=0.731.05]; P=0.158) for chemotherapy alone in the intention to treat populations, and 23.4 versus 10.2 months (HR=0.48 [95% CI=0.290.81]; P not formally calculated) in the PD-L1-high population.77 No differences in median OS were seen between the treatment arms in the overall PD-L1-positive population (14.8 versus 15.0 months), or in PD-L1-negative patients (median 14.0 versus 12.5 months).77

Early-phase studies are also exploring various combinations of approved and investigational agents, including pembrolizumab plus ramucirumab,78 and novel agents such as anlotinib79 and camrelizumab.80

It has been suggested that radiotherapy in addition to chemotherapy plus immune checkpoint inhibitors, the current first-line standard of care for patients with advanced NSCLC, may further improve outcomes, but this strategy is yet to be tested in clinical trials.81

Compared with afatinib monotherapy, afatinib combination therapy with other agents may yield better efficacy results in general EGFR wild-type populations. The Phase II, single-arm LUX-Lung IO/KEYNOTE-497 is investigating the efficacy of afatinib plus pembrolizumab in unselected patients with locally advanced/metastatic squamous cell lung carcinoma that has progressed during or after first-line platinum-based chemotherapy.82 Enrollment for this study has closed, but no results are available as yet.

Agents such as chemotherapy and immune checkpoint inhibitors appear to be the most efficacious therapies across a broad range of patients with squamous cell lung carcinoma when used early in the disease course. Further data are required to establish the optimal place for afatinib within the squamous cell lung cancer treatment landscape. However, until further data are available afatinib may be considered an option for some patients who have progressed on previous therapies but are not eligible for existing, more-established therapies.

Afatinib may be a particularly good second- or third-line option in certain problematic clinical scenarios. When immunotherapy is used alone or in combination with chemotherapy as first-line treatment, the findings from CheckMate 017 (nivolumab versus docetaxel in unselected patients with progressive disease after first-line platinum-based chemotherapy)23 and OAK (atezolizumab versus docetaxel in unselected patients with progressive disease after one or two previous chemotherapy regimens)24 studies cannot be applied. In addition, if the patient was initially treated with pembrolizumab, Keynote-001 (pembrolizumab in patients with treatment failure after prior systemic therapy83) and Keynote-010 (pembrolizumab versus docetaxel in patients with PD-L1 TPS 1% and progressive disease after platinum-containing chemotherapy84) are also not applicable.

These limitations leave only four options as second- or subsequent-line treatment for many patients. These are docetaxel plus ramucirumab, afatinib, gemcitabine as a single agent or as one of several available platinum-doublet chemotherapy options, and participation in a clinical trial. The REVEL trial showed that the combination of docetaxel and ramucirumab was superior to docetaxel alone,22 suggesting that docetaxel monotherapy is no longer appropriate unless the patient cannot receive ramucirumab. As ramucirumab was not studied in patients with centrally-located tumors or cavitation, docetaxel in combination with ramucirumab may not be appropriate in such scenarios.22,85

The second option, oral afatinib monotherapy, has been shown to confer an OS benefit over erlotinib in patients with squamous cell lung cancer.58 Although erlotinib is no longer approved in this indication, and direct comparisons cannot be made with other agents, the OS seen in patients who progressed after platinum-based chemotherapy with afatinib (7.9 months) is comparable to that seen with docetaxel (8.2 months) in the REVEL study in the second-line setting.22 Notably, both the REVEL and LUX-Lung 8 studies were conducted before the immunotherapy era. Recent data among patients with EGFR mutation-positive NSCLC suggesting that the use of afatinib following anti-PD-(L)1 therapy is not associated with severe immune-related adverse events86 are reassuring, and support further investigation of afatinib in patients who have previously received immune checkpoint inhibitors.

The third option is gemcitabine therapy; however, the data supporting its use as a single agent in the second-line setting come primarily from Phase II studies.8789 Certainly, the use of platinum-based doublets incorporating gemcitabine in chemotherapy-nave NSCLC patients is well established, with comparable efficacy to other platinum-based combinations.90 Gemcitabine monotherapy may also be useful in the maintenance setting. In a Phase III study, gemcitabine or erlotinib maintenance was compared with observation alone in patients whose disease was controlled after cisplatin-gemcitabine induction chemotherapy.91 This study demonstrated that maintenance therapy with erlotinib (switch) or gemcitabine (continuation) significantly delayed disease progression after cisplatin-gemcitabine induction.

In summary, afatinib monotherapy may be a suitable therapeutic option for some patients with squamous cell lung cancer in the second- or third-line setting, but further assessment of the optimal place of afatinib within the current treatment landscape is required. Further, biomarker analyses and a small number of case studies suggest that certain groups of patients, such as those harboring mutations in the ErbB family of receptor tyrosine kinases, may derive particular benefit from afatinib. Further studies should help to determine whether efficacy can be improved by the addition of other agents such as pembrolizumab.

Abbreviations

ALK, anaplastic lymphoma kinase; CI, confidence interval; Del19, deletion in exon 19 of the EGFR gene; ECOG, Eastern Cooperative Oncology Group; EGFR, epidermal growth factor receptor; FDA, Food and Drug Administration; HER2, human epidermal growth factor receptor; HR, hazard ratio; NLM, National Library of Medicine; NSCLC, non-small-cell lung cancer; NTRK, neurotrophic receptor tyrosine kinase; OS, overall survival; PD-L1, programmed death-ligand 1; PFS, progression-free survival; SCC, squamous cell carcinoma; TKI, tyrosine kinase inhibitor; TMB, tumor mutational burden; TPS, tumor proportion score; US, United States.

Acknowledgments

The author(s) meet criteria for authorship as recommended by the International Committee of Medical Journal Editors (ICMJE). The authors received no direct compensation related to the development of the Manuscript. Writing, editorial support and formatting assistance was provided by Natalie Grainger and Laura Winton, of GeoMed, an Ashfield company, part of UDG Healthcare plc, which was contracted and funded by Boehringer Ingelheim Pharmaceuticals, Inc. (BIPI). BIPI was given the opportunity to review the Manuscript for medical and scientific accuracy as well as intellectual property considerations.

Disclosure

ES reports speakers bureau fees from Genentech, Astellas, Amgen, Biodesix, Paradigm Diagnostic, Boehringer-Ingelheim, Caris SL, Celgene, Guardant Health, Pfizer, AstraZeneca, Novartis, Merck, Takeda, Sanofi Genzyme, and Dova. He is also consultants for Lilly US Oncology, BluePrint Medicine and Inivata. LH reports grant/research support from Boehringer-Ingelheim, Genentech, Merck, and BMS, consultancy fees from Genentech and Merck, and speakers bureau fees from Genentech and Boehringer Ingelheim. LH also reports he has previously received speakers bureau and/or consulting fees from Novartis and Eli Lilly. He was part of the advisory boards and consultant for G1 Therapeutics. The authors report no other conflicts of interest in this work.

Edgardo S Santos,1 Lowell Hart2,3

1Florida Precision Oncology/A Division of 21st Century Oncology, Florida Atlantic University, Aventura, FL, USA; 2Drug Development Unit, Florida Cancer Specialists, Fort Myers, FL, USA; 3Wake Forest School of Medicine, Winston-Salem, NC, USA

Correspondence: Edgardo S SantosFlorida Precision Oncology/A Division of 21st Century Oncology, Thoracic Oncology, Charles E. Schmidt College of Medicine, Florida Atlantic University, 3585 NE 207th Street, Suite C6-C7, Aventura, FL 33180, USATel +1 561-334-2850Fax +1 305-952-4866Email edgardo.santos@21co.com

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37. Genova C, Socinski MA, Hozak RR, et al. EGFR gene copy number by FISH may predict outcome of necitumumab in squamous lung carcinomas: analysis from the SQUIRE Study. J Thorac Oncol. 2018;13(2):228236. doi:10.1016/j.jtho.2017.11.109

38. Herbst RS, Redman MW, Kim ES, et al. Cetuximab plus carboplatin and paclitaxel with or without bevacizumab versus carboplatin and paclitaxel with or without bevacizumab in advanced NSCLC (SWOG S0819): a randomised, phase 3 study. Lancet Oncol. 2018;19(1):101114. doi:10.1016/S1470-2045(17)30694-0

39. Paz-Ares L, Socinski MA, Shahidi J, et al. Correlation of EGFR-expression with safety and efficacy outcomes in SQUIRE: a randomized, multicenter, open-label, phase III study of gemcitabine-cisplatin plus necitumumab versus gemcitabine-cisplatin alone in the first-line treatment of patients with stage IV squamous non-small-cell lung cancer. Ann Oncol. 2016;27(8):15731579. doi:10.1093/annonc/mdw214

40. Gatzemeier U, Pluzanska A, Szczesna A, et al. Phase III study of erlotinib in combination with cisplatin and gemcitabine in advanced non-small-cell lung cancer: the tarceva lung cancer investigation trial. J Clin Oncol. 2007;25(12):15451552. doi:10.1200/JCO.2005.05.1474

41. Herbst RS, Giaccone G, Schiller JH, et al. Gefitinib in combination with paclitaxel and carboplatin in advanced non-small-cell lung cancer: a phase III trialINTACT 2. J Clin Oncol. 2004;22(5):785794. doi:10.1200/JCO.2004.07.215

42. Herbst RS, Prager D, Hermann R, et al. TRIBUTE: a phase III trial of erlotinib hydrochloride (OSI-774) combined with carboplatin and paclitaxel chemotherapy in advanced non-small-cell lung cancer. J Clin Oncol. 2005;23(25):58925899. doi:10.1200/JCO.2005.02.840

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Advanced Squamous Cell Carcinoma of the Lung: Current Treatment Approaches and the Role of Afatinib - Oncology Nurse Advisor

CSO C. David Pauza, Ph.D. Presents on Cell and Gene Therapy for HIV Disease

Chief Science Officer C. David Pauza, Ph.D. Presents At the Intersection of Genetic Medicine and Immunotherapy: Clinical Experience with a Cell and Gene Therapy for HIV Disease

ROCKVILLE, Md., Oct. 08, 2020 (GLOBE NEWSWIRE) -- AmericanGene Technologies(AGT)a cutting-edge cell and gene therapy company in Rockville, Maryland announced today that CEO Jeff Galvin will present at the annual Cell & Gene Meeting on the Mesa. The meeting will be held virtually October 12-16. Galvins presentation will highlight the company's technology, including AGT103-T, a therapeutic intended to cure HIV, which is scheduled to begin Phase 1 clinical trials this month.

Specifics of AGTs clinical trial can be found at https://clinicaltrials.gov/ct2/show/NCT04561258, and details of the HIV therapy intended to cure the disease are on the AGT web site.

Organized by the Alliance for Regenerative Medicine, the Cell & Gene Meeting on the Mesa is a five-day virtual conference featuring more than 120 presentations from the leading public and private companies. These presentations will highlight the most exciting technical and clinical achievements from the past 12 months in cell therapy, gene therapy, gene editing, tissue engineering, and broader regenerative medicine technologies. The meeting also includes over 100 panelists and features speakers taking part in 20 in-depth sessions covering all aspects of cell and gene therapy commercialization.

The following are specific details regarding AmericanGene Technologies presentation at the conference:

Please visit http://www.meetingonthemesa.com for full information including registration. Complimentary attendance at this event is available for accredited investors and members of the media. Investors should contact Laura Stringham at lstringham@alliancerm.org and interested media should contact Kaitlyn Dupont at kdupont@alliancerm.org. The event hashtag is #CGMOM20.

About American Gene Technologies (AGT)

AmericanGene Technologies(AGT)is a gene and cell therapy company with a proprietary gene-delivery platform for rapid development of cell and gene therapies. AGTs mission is to transform peoples lives by designing highly effective therapeutics to cure infectious diseases, cancers, and inherited disorders. AGT has received three patents for the technology used to make the AGT103-T cell product and ten patents for its uniqueimmuno-oncology approachto stimulategamma-delta () T cellsto destroy a variety of solid tumors. The company has also developed a synthetic gene for treating Phenylketonuria (PKU), a debilitating inherited disease. AGT's treatment for PKU has been grantedOrphan Drug Designationby the Food and Drug Administration (FDA), and it is expected to reach the clinic in 2021.

More information is available on:

Website - http://www.americangene.comLinkedIn - LinkedInTwitter -@americangeneFacebook -@amerigeneInstagram -@americangenetechnologies

American Gene Technologies Contacts:

C. Neil Lyons, Chief Financial OfficerPhone: (301) 337-2269Email:info@americangene.comwww.americangene.com

Sasha Whitaker, Digital Marketing and CommunicationsPhone: (301) 337-2100Email:swhitaker@americangene.comwww.americangene.com

A video is available at the following link:https://www.globenewswire.com/NewsRoom/AttachmentNg/2623868c-a33f-49c1-bc46-0c114cbd1d83

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American Gene Technologies to Present at 2020 Virtual Cell & Gene Meeting on the Mesa - GlobeNewswire

This report describes and evaluates the global nucleic acid-based gene therapy market. It covers three five-year periods, including 2015 to -2020, termed the historic period, 2020-2025 forecast period and 2025-2030 a further forecast period.

LONDON, Oct. 05, 2021 (GLOBE NEWSWIRE) -- According to The Business Research Companys research report on the nucleic acid-based gene therapy market, companies in the nucleic acid-based gene therapy market and research institutes are increasing the number of pipeline studies to develop gene therapy to treat various diseases. Companies have also started investing in startups and other early-stage companies to develop pipelines for gene therapies. Cell and gene therapies (CGT) have transformed not only how humans treat intractable and genetic diseases, but also reformed the entire pharmaceutical ecosystem. As of 2019, more than 27 CGT products were launched across the globe and nearly 990 companies are engaged in the commercialization, and research & development of next-generation therapies. Additionally, there are more than 1,000 regenerative medicine trials taking place across the globe.

Such global nucleic acid based gene therapy market trends are obtainable with nucleic acid-based gene therapy manufacturers progressively investing in the launch of new manufacturing facilities and product portfolio expansion to meet the increasing demand for gene therapy and related products. Players operating in the nucleic acid-based gene therapy market are gradually investing in the developing regions to capitalize on untapped market opportunities. For example, in September 2021, Viralgen, a Bayer-owned CDMO, spent upwards of 50 million (US$ 55 million) to expand its capacity for gene therapy manufacturing services at its Miramon Technology Park site in San Sebastian, Spain. The commercial facility will have nine cleanrooms, each with a batch capacity of up to 2,000 L. Viralgen claims that this has expanded its existing viral vector capacity 15-fold, helping to meet the demand for gene therapy production. In addition, in May 2021, AGC Biologics, a global biopharmaceutical contract development and manufacturing organization (CDMO), announced plans to expand their Gene Therapy Center of Excellence in Milan, Italy.

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Major players in the nucleic acid gene therapy market include Copernicus Therapeutics, Moderna Inc., Wave Life Sciences, Protagonist Therapeutics and Transgene.

The Business Research Companys report titled Nucleic Acid Based Gene Therapy Global Market Report 2021 - By Technology (Anti-Sence and Anti-Gene, Short Inhibitory Sequences, Gene Transfer Therapy, Nucleoside Analogs, Ribozymes, Aptamers), By Application (Oncology, Muscular Dystrophy/ Muscular Disorders, Rare Diseases), By End User (Hospitals And Clinics, Academic And Research Institutes), COVID-19 Growth And Change covers major nucleic acid based gene therapy companies, nucleic acid based gene therapy market share by company, nucleic acid based gene therapy manufacturers, nucleic acid based gene therapy market size, and nucleic acid based gene therapy market forecasts. The report also covers the global nucleic acid based gene therapy market and its segments.

Request For A Sample Of The Global Nucleic Acid Based Gene Therapy Market Report:

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The global nucleic acid based gene therapy market size is expected to grow from $0.56 billion in 2020 to $0.61 billion in 2021 at a compound annual growth rate (CAGR) of 8.9%. The growth is mainly due to the companies resuming their operations and adapting to the new normal while recovering from the COVID-19 impact, which had earlier led to restrictive containment measures involving social distancing, remote working, and the closure of commercial activities that resulted in operational challenges. The nucleic acid-based gene therapy market is expected to reach $0.85 billion in 2025 at a CAGR of 9%.

North America is the largest region in the global nucleic acid-based gene therapy market, accounting for 46.2% of the total in 2020. It is followed by the Western Europe, Asia Pacific and then the other regions. Going forward, the fastest-growing regions in the nucleic acid-based gene therapy market will be the Middle East and Eastern Europe where growth will be at CAGRs of 33.7% and 26.0% respectively. These will be followed by South America and Asia Pacific, where the markets are expected to register CAGRs of 21.0% and 20.4% respectively.

The nucleic acid-based gene therapy market covered in this report is segmented by technology into anti-sense and anti-gene, short inhibitory sequences, gene transfer therapy, nucleoside analogs, ribozymes, aptamers, others. It is also segmented by application into oncology, muscular dystrophy/ muscular disorders, rare diseases and by end user into hospitals and clinics, academic and research institutes.

The top opportunities in the nucleic acid-based gene therapy market segmented by technology will arise in the anti-sense and anti-gene oligonucleotides segment, which will gain $1,290.7 million of global annual sales by 2025. The top opportunities segmented by application will arise in the muscular dystrophy/muscular disorders segment, which will gain $1,000.2 million of global annual sales by 2025, segmented by end-user will arise in the hospitals and clinics segment, which will gain $2,133.7 million of global annual sales by 2025. The nucleic acid-based gene therapy market size will gain the most in the USA at $915.0 million.

Nucleic Acid Based Gene Therapy Global Market Report 2021 COVID-19 Growth And Change is one of a series of new reports from The Business Research Company that provide nucleic acid-based gene therapy market overviews, nucleic acid-based gene therapy market analyze and forecast market size and growth for the whole market, nucleic acid-based gene therapy market segments and geographies, nucleic acid-based gene therapy market trends, nucleic acid-based gene therapy market drivers, nucleic acid-based gene therapy market restraints, nucleic acid-based gene therapy market leading competitors revenues, profiles and market shares in over 1,000 industry reports, covering over 2,500 market segments and 60 geographies.

The report also gives in-depth analysis of the impact of COVID-19 on the market. The reports draw on 150,000 datasets, extensive secondary research, and exclusive insights from interviews with industry leaders. A highly experienced and expert team of analysts and modelers provides market analysis and forecasts. The reports identify top countries and segments for opportunities and strategies based on market trends and leading competitors approaches.

Here Is A List Of Similar Reports By The Business Research Company:

Gene Editing Global Market Report 2021 - By Technology (CRISPR, TALEN, ZFN), By End Users (Biotechnology, Pharmaceutical, Contract Research Organization), By Application (Animal Genetic Engineering, Plant Genetic Engineering, Cell Line Engineering), COVID-19 Growth And Change

CRISPR Technology Global Market Report 2021 - By Product Type (Design Tools, Plasmid And Vector, Cas9 And G-RNA, Delivery System Products), By Application (Genome Editing/ Genetic Engineering, Genetically Modified Organisms, Agricultural Biotechnology), By End-User (Industrial Biotech, Biological Research, Agricultural Research, Therapeutics And Drug Discovery), COVID-19 Growth And Change

Stem Cell Therapy Global Market Report 2021 - By Type (Allogeneic Stem Cell Therapy, Autologous Stem Cell Therapy), By Cell Source (Adult Stem Cells, Induced Pluripotent Stem Cells, Embryonic Stem Cells), By Application (Musculoskeletal Disorders, Wounds And Injuries, Cancer, Autoimmune Disorders), By End-User (Hospitals, Clinics), COVID-19 Growth And Change

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Nucleic Acid Based Gene Therapy Market Analysis Of Industry Trends And Market Growth Opportunities As Per The Business Research Company's Nucleic Acid...

Introduction

Colorectal cancer (CRC) has become the predominant cancer worldwide with more than 1.8 million new cases diagnosed annually.1,2 Furthermore, the five-year survival rate for patients with advanced-stage metastatic cancer is approximately 10%.1 Like other cancers, CRC is considered to be a heterogeneous disease in which gene aberrations, cellular context, and environmental influences concur with tumor initiation, progression, and metastasis.3 Despite advances in laparoscopic and robotic surgery, more aggressive resection of metastatic disease, radiotherapy, as well as neoadjuvant and palliative chemotherapies, the new treatments had an insignificant effect on long-term survival.4 Thus, it is critical to make a thorough inquiry into the underlying biological mechanism of the occurrences and metastases of cancers associated with prognosis so as to discover novel biomarkers for target therapies and prognosis predictions. Although accumulating evidence has demonstrated that multiple genes and cellular pathways participate in the occurrence and development of CRC,5,6 a paucity of knowledge regarding the potential precise molecules and potential mechanisms underlying CRC progression currently limits the ability to treat this disease.

Bioinformatics analyses, including the analysis of gene interaction networks, microarray expression profiles, and gene annotations are being utilized as powerful tools for identifying potential diagnostic and treatment-relevant biomarkers of cancers.7,8 For example, by analyzing data from the Gene Expression Omnibus (GEO) database, Cao et al9 identified five genes as potential biomarkers and therapeutic targets for gastric cancer. In addition, by analyzing data from GEO and The Cancer Genome Atlas (TCGA), Zhu et al found that high expression of cyclin-dependent kinase 1 (CDK1) is a prognostic factor for hepatocellular carcinoma (HCC), making it a potential therapeutic target and biomarker for the diagnosis of HCC.10 In particular, the method of integrated bioinformatics analysis can be used to overcome inaccuracies in sequencing arising from small sample sizes. Circular RNAs (circRNAs) are a novel class of endogenous non-coding RNAs that form a covalently closed-continuous loop by back-splicing events via exon or intron circularization.11 Due to the development of high-throughput sequencing, researchers have discovered that thousands of circRNAs are involved in the progression of oncogenesis, invasion, and metastasis by playing the role of sponges to microRNAs (miRNAs).12 For instance, Wang et al13 verified that circDLGAP4 regulated lung cancer cell biological processes by sponging miRNA-143 to regulate CDK1 expression and circDLGAP4 may serve as a potential biomarker for the diagnosis and treatment of lung cancer. However, at present, most studies involving circRNAs have been limited to the sequencing of a few samples or exploring the biological function of single circRNAs. To the best of our knowledge, currently, no researchers have used integrated analysis to investigate CRC-related circRNAs.

In this study, differentially expressed mRNAs (DEmRNAs) between human CRC tissues and adjacent non-tumor tissues were identified via analysis of public TCGA datasets. Next, to explore the roles of these DEmRNAs, functional enrichment analyses and pathway enrichment analyses were performed. Then, proteinprotein interaction (PPI) networks were successfully constructed. The key genes and significant modules in the networks were identified. KaplanMeier analysis was performed to evaluate the prognostic value of these hub genes. Furthermore, three additional circRNA expression profiles were analyzed to identify differentially expressed circRNAs (DEcircRNAs) and differentially expressed miRNAs (DEmiRNAs) between CRC and adjacent non-tumor tissues. Finally, circRNAsmiRNAsmRNA ceRNAs network was constructed. The research is expected to help to further elucidate the ceRNAs interactions in CRC and generate insight into the potential biomarkers and targets for the diagnosis, prognosis, and therapy of CRC.

CRC gene expression profile data were downloaded from TCGA (https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga) and standardized, including 41 normal samples and 473 tumor samples and their clinical information. Previous studies have demonstrated that without adjustment, TCGA-COAD READ data set could be generated by merging samples from TCGA-COAD data set and TCGA-READ data set, since principal components analyses and unsupervised hierarchical clustering showed no significant differences.14,15 CRC miRNA expression profiles from Illumina HiSeqmiRNASeq platforms, including 8 normal samples and 450 tumor samples, were downloaded from TCGA and standardized. In addition, 4 circRNA expression profiles (GSE121895, GSE126094, GSE138589, GSE142837) from Illumina HiSeqRNASeq platforms, including 23 tumor samples and 23 normal samples, were downloaded from GEO (http://www.ncbi.nlm.nih.gov/geo) by searching for the term CRC (July 2020), and batch effects were removed using the combat function in the R sva package.16

DEcircRNAs, DEmRNAs, and DEmiRNAs were identified using an R package DESeq2 (http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html). |log2FC| >2 and FDR <0.05 were set as the cutoff criteria (FC, fold change; FDR, false discovery rate) based on the BenjaminiHochberg method for DEmRNAs and DEmiRNAs.17 DEcircRNAs were screened by |log2FC| >1 and FDR<0.05. R was used to visualize differential genes. For DEcircRNAs, we used Surrogate Variable Analysis to handle multiple GSE profiles as described above. Volcano maps were plotted based on the volcano map of R.

To identify the biological function of the ceRNAs network, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses are widely used for gene annotation terms and pathway enrichment analysis. GO is a widely used tool for annotating genes with functions, especially molecular function (MF), biological pathways (BP), and cellular components (CC). KEGG Enrichment Analysis is a practical resource for the analytical study of gene functions and associated high-level genome functional information. ClusterProfiler package of R was performed to analyze and visualize functional profiles. A P-value < 0.05 was the threshold for significance for GO and KEGG terms.

The PPI network was conducted to analyze the functional interactions between proteins, providing insights into the mechanisms for the development of CRC. The minimum required interaction score is 0.5. The STRING website (https://string-db.org/) was employed to construct the PPI network.

Based on the median expression level of each DEmRNAs, the CRC patients were divided into high and low-expression groups. KaplanMeier analysis and the Log rank test were utilized to paint the survival curves to find the DEmRNAs that were significantly associated with the survival of CRC patients. A P-value < 0.05 was set as the threshold.

DEcircRNAs and DEmRNA matched by DEmiRNAs were retrieved using starBase database.18 Moreover, the prediction results of TargetScan, miRTarBase, and miRDB were integrated by starBase.1921 The candidates searched in three databases were associated with the most important DEmiRNAs. Finally, the circRNAsmiRNAsmRNA ceRNAs network was constructed and visualized using R.

Total RNA was prepared from colonic tissue using an RNA extraction kit (TIANGEN BIOTECH, Beijing, China), according to the manufacturers instructions. The extracted RNA was synthesized to form cDNA using a FastKing one-step kit (TIANGEN BIOTECH, Beijing, China). qRT-PCR was performed using a RealUniversal Color PreMix (SYBR Green) kit (TIANGEN BIOTECH, Beijing, China) to assess the expression of target genes. U6 was used as an internal control for DEmiRNAs. GAPDH was used as internal control for TIMP1. In addition, the relative expression of RNAs was quantified by using the 2Ct method.

Table 1 shows the clinicopathological data of 473 patients with CRC. According to the cutoff threshold, a total of 412 DEmRNAs (including 82 upregulated and 330 downregulated) were screened out between 473 CRC and 41 normal samples with the standard of logFC> 2 and an adjusted P value (adj.P.Val) <0.05 (Figure 1A). Two hundred and sixty DEcircRNAs (including 253 upregulated and 7 downregulated) were altered significantly between 23 CRC and 23 normal samples by log2FC > 1 and an adj.P.Val < 0.05 (Figure 1B). To further establish an circRNAsmiRNAsmRNAs ceRNAs network, we also matched DEmiRNA expression profiles in the 450 CRC and 8 normal samples. As a result, 190 DEmiRNAs reached the inclusion criteria including 82 upregulated and 108 downregulated miRNAs (Figure 1C). The top 10 DEcircRNAs, DEmiRNAs, and DEmRNAs are presented in Table 2.

Table 1 Clinicopathological Characteristics of 473 CRC Patients

Table 2 Top 10 DEcircRNAs, DEmiRNAs and DEGs in Human CRC

Figure 1 The volcano maps of DEGs between CRC samples and normal samples. (A) A total of 412 DEmRNAs including 82 upregulated and 330 downregulated genes. (B) A total of 260 DEcircRNAs including 253 upregulated and 7 downregulated genes. (C) A total of 190 DEmiRNAs including 82 upregulated and 108 downregulated genes. Red represents upregulated genes and green represents downregulated genes.

To further analyze the functional characteristics of DEmRNAs in CRC, GO and KEGG pathway analyses were performed using ClusterProfiler package of R. DEmRNAs were functionally classified into the biological process (BP), cellular component (CC), and molecular function (MF categories). In the BP category, four of the nine most enriched terms were regulation of protein processing, protein activation cascade, regulation of acute inflammatory response and complement activation. In the CC category, the four most enriched terms were extracellular matrix, collagen-containing extracellular matrix, blood microparticle and apical part of cell. In the MF categories, the three most enriched terms were antigen binding, receptor regulator activity and receptor ligand activity (Figure 2A). In addition, almost 16 KEGG pathways were significantly enriched in our analysis. The three most enriched terms were cytokine-cytokine receptor interaction, kineral absorption and steroid hormone biosynthesis (Figure 2B).

Figure 2 Functional enrichment analysis of DEmRNAs. (A) The top 9 enrichment scores in GO enrichment analysis of the DEmRNAs including biological process enrichment analysis, cellular components enrichment analysis, molecular function enrichment analysis. (B) The top 16 enrichment scores in KEGG enrichment analysis of the DEmRNAs.

A total of 412 DEmRNAs (82 upregulated and 330 downregulated) were used to construct the PPI networks, which included 226 nodes and 478 edges. The combined minimum required interaction score >0.5 was considered statistically significant (Figure 3). In addition, the degree distribution of each gene in the PPI network was analyzed, the top five genes [C-X-C chemokine receptor type 8 (CXCL8), TIMP1 (tissue inhibitor of metalloproteinase 1), CXCL1, secreted phosphoprotein 1 (SPP1) and CXCL12] with high connectivity were confirmed as hub genes and next were underwent survival analysis.

Figure 3 The plot of the PPI network of DEmRNAs including 226 nodes and 478 edges by the online database STRING. The combined minimum required interaction score>0.5 was considered statistically significant.

The prognostic values of the five hub genes were assessed in CRC patients using KaplanMeier analysis and Log rank test. The results indicated that CRC patients with high expression of TIMP1 showed worse overall survival (P=0.004). In contrast, the other four hub genes (CXCL8, CXCL1, SPP1, and CXCL12) were not related to the overall survival of CRC patients (P > 0.05) (Figure 4).

Figure 4 Kaplan-Meier survival curves for the top five hub genes including SPP1, CXCL1, TIMP1, CXCL8, and CXCL12. TIMP1 was significantly associated with survival rate of CRC patients.

miRNAs-mRNA interactivity was taken into account, in addition to the circRNAs-miRNAs, to construct an integrated ceRNAs network. Based on the starBase database, which masters the function of transcriptome-wide mircoRNA targeting prediction, we matched 61 DEcircRNAs and 3 DEmiRNAs. To clearly show the interaction in ceRNAs, the regulatory network contained some well-described biomarkers, including, hsa-miR-671-5p, hsa-miR-17-3p, hsa-miR-328-3p and TIMP1. This ceRNAs network is particularly informative in locating potential biomarkers for CRC. For instance, hsa-miR-671-5p interacted with TIMP1 and was mediated by hsa-circ-0002191. hsa-miR-17-3p interacted with TIMP1 and was mediated by has-circ-0023397 (Figure 5).

Figure 5 The ceRNAs network of circRNAs-miRNAs-mRNA in CRC. Blue represents DEcircRNAs; Black represents DEmiRNAs; Red represents DEmRNA.

To identify the authenticity and feasibility of the ceRNAs regulatory network, some vital DEmiRNAs and DEmRNAs are evaluated in colon cancer tissue and normal tissues. We found that TIMP1 is highly expressed in colon cancer tissue compared to normal tissue (P < 0.001). In contrast, the expression levels of hsa-miR-671-5p, hsa-miR-17-3p, and hsa-miR-328-3p were significantly decreased in colon cancer tissue (Figure 6).

Figure 6 The expression levels of DEmRNA and DEmiRNAs in colon cancer patients compared with those of normal samples. (A) The TIMP1 is highly expressed in colon cancer tissue. (BD) The miR-671-5p, miR-17-3p and miR-328-3p is low expression in colon cancer tissue, **P <0.01, and ***P <0.001.

CRC, the third most commonly diagnosed malignancy and the second leading cause of cancer-related deaths with notably aggressive biological behavior and poor survival rates, has always drawn close attention from researchers.2 It is crucial to identify reliable therapeutic targets and biomarkers in order to improve the clinical outcome for CRC patients. The ceRNAs hypothesis presents a new pattern of gene expression regulation that cicrRNAs could regulate mRNAs by competing with the corresponding miRNAs.22 Subsequently, benefits from developments in sequencing technology and the applications of bioinformatics confirm the increasingly important biological role in the initiation, progression, and metastasis of tumors.1921 CircRNAs differ from other long non-coding RNAs in the structure, which is characterized by covalently linked 5- and 3-ends. CircRNAs functionally act as miRNAs sponges, RNA-binding protein sponges, and gene expression regulators. Therefore, circRNAs regulate their target genes expression and proteins network in both transcriptional and post-transcriptional patterns.23 Increasingly, clinicians consider that circRNAs-miRNAs-mRNAs ceRNAs networks could provide an integrated view of regulatory crosstalk between these CRC-specific RNA transcripts.24,25

In this study, DEmRNAs were identified between tumor samples and normal control tissues. Then, GO and KEGG analyses were performed to further understand the role of DEmRNAs. The results of GO analyses showed that the DEmRNAs were enriched in regulation of protein processing, protein activation cascade, and acute inflammatory which is confirmed by the knowledge that protein-induced pathology and inflammatory networks underlying CRC are the main cause for tumor development and progression.2628 Furthermore, KEGG analyses showed that cytokinecytokine receptor interaction is a substantial factor in the occurrence of CRC. Cytokines such as TNF- and IL-6 are classically regarded as central players in CRC by driving activation of the NF-B and STAT329. Cytokines including IL-11, IL-17A, and IL-22 have gained attention as facilitators of CRC.29

The top degree hub genes (CXCL8, TIMP1, CXCL1, SPP1 and CXCL12) were presented in the PPI network with DEmRNAs. SPP1, also named Osteopontin, has been proven to be overexpressed in various malignant neoplasms including breast cancer, lung cancer, and gastric cancer.3032 Although Seo et al33 have evaluated the expression of SPP1 in 174 stage II and III CRC specimens and found SPP1 is significantly associated with cell invasion and adherence in CRC, the underlying mechanism was not revealed. Wang et al34 has shown that SPP1 functions as an enhancer of cell growth in hepatocellular carcinoma (HCC) targeted by miR-181c. Further studies have shown that SPP1 promotes the metastasis of CRC by activating epithelial-mesenchymal transition (EMT).35 CXCL8, as a prototypical chemokine, is responsible for the recruitment and activation of neutrophils and granulocytes to the site of inflammation which demonstrated that CXCL8 played a crucial role in facilitating tumor growth and progression in breast cancer, prostate cancer, lung cancer, colorectal carcinoma, and melanoma.36 Phosphorylation of Src-kinases and focal adhesion kinase (FAK) in cancer cells were increased in CXCL8 signaling, which contributed to cell proliferation and chemoresistance.37,38 The level of CXCL1 are elevated in CRC and increased level of CXCL1 are associated with tumor size, advancing stage, and patient survival.39,40 It was reported that CXCL1 could promote tumor growth by inducing angiogenesis and the recruitment of neutrophils into the tumor-associated microenvironment.41,42 CXCL1, the most abundant secreted chemokine by tumor-associated macrophages has been implicated in the promotion of breast cancer growth and metastasis via activating NF-B/SOX4 signaling.43 The similar phenomenon has been observed in human bladder cancer.44 Some researchers have indicated that CXCL1 could increase oncogenes expression in colon cancer, including forkhead box O1 (FOXO1) and transcription factor 4 (TCF4) in CXCL1-treated SW620 cells according to transcriptome analyses.45 CXCL1 is also vital for pre-metastatic niche formation and metastasis in CRC.46 CXCL12 also known as SDF-1 is widely distributed in human tissues and more than 23 different types of cancers.47 Importantly, it has been found that CXCL4 and its ligand CXCL12 are implicated in cell proliferation, angiogenesis, migration, EMT, and tumor metastasis.48 TIMP1 belongs to the tissue inhibitor of the metalloproteinases family which includes TIMP1, TIMP2, TIMP3, and TIMP4.49 In the present study, TIMP1 has been reported to indicate poor prognosis in CRC (P=0.004), which is consistent with the research of Song et al.50 Song et al considers that the expression of TIMP1 was clearly associated with the regional lymph node and distant metastasis. In addition, research by Song et al indicated that TIMP1 was an independent prognostic indicator for the progression and metastasis of colon cancer through FAK-PI3K/AKT and MAPK pathway.50 Moreover, TIMP1 could promote receptor tyrosine kinase c-Kit signaling in CRC, while c-Kit is an important oncogene in CRC and plays a role in cell proliferation and migration.51 For other cancers, TIMP1 inhibited the chemosensitivity of breast cancer cells through the PI3K/AKT/NF-kB pathway.52 TIMP1 is in favor of cell survival in melanoma by activating the 3-phosphoinositide dependent kinase-1 signaling pathway.53 TIMP1/CD63/ERK signaling axis induces the formation of neutrophil extracellular traps and facilitates the development of pancreatic cancer.54 Clinical studies have demonstrated that the elevated level of TIMP1 was associated with poor prognosis in various tumors, such as breast cancer,55 cutaneous melanoma,56 and gastric cancer.57 The elevated plasma level of TIMP1 predicted a reduced response to second-line hormone therapy and low survival in women with metastatic breast cancer.58 Therefore, TIMP1 may be a potential biomarker to predict the prognosis of cancer and play a critical role as a therapeutic target. The TIMP-miRNAs axis has been believed to be a potential therapeutic target against aggressive or drug-resistant variants of human cancers.5961 For instance, angiogenesis and tumor growth were increased when TIMP1 banded to CD63 and stimulated miR-210 accumulation by activating PI3KAKTHIF1 signaling in the lung adenocarcinoma.60 As the hub elements of the ceRNAs network, miRNAs exhibited key roles among different RNA transcripts. In fact, hsa-miR-671-5p have been proven to interact with TIMP1 directly by cross-linking immunoprecipitation.62 However, the interaction between hsa-miR-671-5p and TIMP1 still needs to be verified in the occurrence and progress of CRC. The miR-671-5p had a protective role in gastric cancer by targeting upregulator of cell proliferation.63 Meanwhile, miR-671-5p inhibits EMT by directly downregulating FOXM1 in breast cancer.64 Interestingly, the levels of miR-671-5p are not only increased in colon cancer tissue but also increased cell proliferation, migration, and invasion by targeting tripartite motif containing 67.65 This finding runs against our findings. The same miRNAs can regulate multiple mRNAs molecules and produce different physiological effects. MiR-328-3p was identified in bladder cancer and suppressed cell proliferation, migration, and invasion by targeting integrin subunit alpha 5 as well as by inhibiting EMT and inactivated PI3K/AKT pathway.66 Similar tumor suppression effects were observed in colon cancer.67

The present study identifies a novel ceRNAs network, which implies that TIMP1 is a potential biomarker underlying the development of CRC, providing new insights into survival predictions and therapeutic targets. However, the limitation of the study still needs investigation. Too many circRNAs were chosen in this study, a more advanced approach to narrow the scope of research is needed. The results of the present CRC-related ceRNAs regulatory network are required to be verified by clinical trials and molecular experiments.

The present study identified a novel circRNAs-miRNAs-mRNAceRNAs network and provided candidate prognostic biomarkers for predicting the outcome of patients with CRC. Especially, TIMP1 is a potential indicator underlying the development of CRC. This study provided new insights for the survival predictions and therapeutic targets of CRC.

All the experiments were approved by the Ethics Committee of Tianjin Medical University General Hospital (Tianjin, China).

All authors contributed to data analysis, drafting or revising the article, gave final approval for the version to be published, agreed to the submitted journal, and agreed to be accountable for all aspects of the work. Ya-Fei Qin, Guang-Ming Li and Grace Wang are co-first authors of this paper.

This work was supported by grants to Hao Wang from the National Natural Science Foundation of China (No. 82071802), Tianjin Application Basis and Cutting-Edge Technology Research Grant (No. 14JCZDJC35700), Li Jieshou Intestinal Barrier Research Special Fund (No. LJS_201412), Natural Science Foundation of Tianjin (No. 18JCZDJC35800), and Tianjin Medical University Talent Fund; by a grant to Dejun Kong from Tianjin Research Innovation Project for postgraduate students (No. 2019YJSS184).

The authors declare that they have no competing interests.

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ceRNAs network in the pathophysiological development of CRC | TCRM - Dove Medical Press

NEW YORK and CLEVELAND, Dec. 21, 2020 (GLOBE NEWSWIRE) -- Abeona Therapeutics Inc. (Nasdaq: ABEO), a fully-integrated leader in gene and cell therapy, today announced that abstracts detailing new interim results from its ABO-102 Phase 1/2 Transpher A study for MPS IIIA and ABO-101 Phase 1/2 Transpher B study for MPS IIIB have been accepted for platform oral presentations during the late-breaking abstract session at the 17th Annual WORLDSymposium being held February 8-12, 2021.

Children born with MPS IIIA and MPS IIIB experience progressive neurodevelopmental decline and loss of motor function that is life-threatening, said Michael Amoroso, Chief Operating Officer of Abeona. We are excited to share new analyses from the Transpher A study that will add to the understanding of the potential for ABO-102 to help preserve neurocognitive development in patients with MPS IIIA when they are treated at a young age, and new results from the Transpher B study that will provide insights into ABO-101s biologic effect in patients with MPS IIIB.

Presentation Details

Title: Updated Results of Transpher A, a Multicenter, Single-Dose, Phase 1/2 Clinical Trial of ABO-102 Gene Therapy for Sanfilippo Syndrome Type A (Mucopolysaccharidosis IIIA)Abstract Number: 390Presenter: Kevin Flanigan, M.D., Center for Gene Therapy at Nationwide Childrens HospitalDate/Time: Friday, February 12, 2021, time to be determined

Title: Updated Results of Transpher B, a Multicenter, Single-Dose, Phase 1/2 Clinical Trial of ABO-101 Gene Therapy for Sanfilippo Syndrome Type B (Mucopolysaccharidosis IIIB)Abstract Number: 407Presenter: Maria Jose de Castro, M.D., Hospital Clnico Universitario Santiago de CompostelaDate/Time: Friday, February 12, 2021, time to be determined

About the Annual WORLDSymposium The WORLDSymposium is designed for basic, translational and clinical researchers, patient advocacy groups, clinicians, and all others who are interested in learning more about the latest discoveries related to lysosomal diseases and the clinical investigation of these advances. For additional information on the 17th Annual WORLDSymposium, please visit https://worldsymposia.org/.

About the Transpher A Study The Transpher A Study (NCT02716246) is an ongoing, two-year, open-label, dose-escalation, Phase 1/2 global clinical trial assessing ABO-102 for the treatment of patients with Sanfilippo syndrome type A (MPS IIIA). The study, also known as ABT-001, is intended for patients from birth to 2 years of age, or patients older than 2 years with a cognitive developmental quotient of 60% or above. ABO-102 gene therapy is delivered using AAV9 technology via a single-dose intravenous infusion. The study primary endpoints are neurodevelopment changes and safety, with secondary endpoints including behavior evaluations, quality of life, enzyme activity in cerebrospinal fluid (CSF) and plasma, heparan sulfate levels in CSF, plasma and urine, and brain and liver volume.

About the Transpher B Study The Transpher B Study (NCT03315182) is an ongoing, two-year, open-label, dose-escalation, Phase 1/2 global clinical trial assessing ABO-101 for the treatment of patients with Sanfilippo syndrome type B (MPS IIIB). The study, also known as ABT-002, is intended for patients from birth to 2 years of age, or patients older than 2 years with a cognitive developmental quotient of 60% or above. ABO-101 gene therapy is delivered using AAV9 technology via a single-dose intravenous infusion. The study primary endpoints are neurodevelopment changes and safety, with secondary endpoints including behavior evaluations, quality of life, enzyme activity in cerebrospinal fluid (CSF) and plasma, heparan sulfate levels in CSF, plasma and urine, and brain and liver volume.

About ABO-102 ABO-102 is a novel gene therapy in Phase 1/2 development for Sanfilippo syndrome type A (MPS IIIA), a rare lysosomal storage disease with no approved treatment that primarily affects the central nervous system (CNS). ABO-102 is dosed in a one-time intravenous infusion using a self-complementary AAV9 vector to deliver a functional copy of the SGSH gene to cells of the CNS and peripheral organs. The therapy is designed to address the underlying SGSH enzyme deficiency responsible for abnormal accumulation of glycosaminoglycans in the brain and throughout the body that results in progressive cell damage and neurodevelopmental and physical decline. In the U.S., Abeona holds Regenerative Medicine Advanced Therapy, Fast Track, Rare Pediatric Disease, and Orphan Drug designations for the ABO-102 clinical program. In the EU, the Company holds PRIME and Orphan medicinal product designations.

About ABO-101 ABO-101 is a novel gene therapy in Phase 1/2 development for Sanfilippo syndrome type B (MPS IIIB), a rare lysosomal storage disease with no approved therapy that primarily affects the central nervous system (CNS). ABO-101 is dosed in a one-time intravenous infusion using a self-complementary AAV9 vector to deliver a functional copy of the NAGLU gene to cells of the CNS and peripheral tissues. The therapy is designed to address the underlying NAGLU enzyme deficiency responsible for abnormal accumulation of glycosaminoglycans in the brain and throughout the body that results in progressive cell damage and neurodevelopmental and physical decline. In the U.S., Abeona holds Fast Track and Rare Pediatric Disease designations for ABO-101 and Orphan Drug designation in both the U.S. and EU.

About Sanfilippo Syndrome Type A (MPS IIIA) Sanfilippo syndrome type A (MPS IIIA) is a rare, fatal lysosomal storage disease with no approved treatment that primarily affects the CNS and is characterized by rapid neurodevelopmental and physical decline. Children with MPS IIIA present with progressive language and cognitive decline and behavioral abnormalities. Other symptoms include sleep problems and frequent ear infections. Additionally, distinctive facial features with thick eyebrows or a unibrow, full lips and excessive body hair for ones age, and liver/spleen enlargement are also present in early childhood. MPS IIIA is caused by genetic mutations that lead to a deficiency in the SGSH enzyme responsible for breaking down glycosaminoglycans, which accumulate in cells throughout the body resulting in rapid health decline associated with the disorder.

About Sanfilippo syndrome type B (MPS IIIB) Sanfilippo syndrome type B (MPS IIIB) is a rare and fatal lysosomal storage disease with no approved therapy that primarily affects the central nervous system and is characterized by rapid neurodevelopmental and physical decline. Children with MPS IIIB present with progressive language and cognitive decline and behavioral abnormalities. Other symptoms include sleep problems and frequent ear infections. Additionally, distinctive signs such as facial features with thick eyebrows or a unibrow, full lips and excessive body hair for ones age and liver/spleen enlargement are also present. The underlying cause of MPS IIIB is a deficiency in the NAGLU enzyme responsible for breaking down glycosaminoglycans, which accumulate throughout the body resulting in rapid decline associated with the disorder.

About Abeona Therapeutics Abeona Therapeutics Inc. is a clinical-stage biopharmaceutical company developing gene and cell therapies for serious diseases. Abeonas clinical programs include EB-101, its autologous, gene-corrected cell therapy for recessive dystrophic epidermolysis bullosa in Phase 3 development, as well as ABO-102 and ABO-101, novel AAV-based gene therapies for Sanfilippo syndrome types A and B (MPS IIIA and MPS IIIB), respectively, in Phase 1/2 development. The Companys portfolio also features AAV-based gene therapies for ophthalmic diseases with high unmet medical needs. Abeonas novel, next-generation AIM capsids have shown potential to improve tropism profiles for a variety of devastating diseases. Abeonas fully functional, gene and cell therapy GMP manufacturing facility produces EB-101 for the pivotal Phase 3 VIITAL study and is capable of clinical and commercial production of AAV-based gene therapies. For more information, visit http://www.abeonatherapeutics.com.

Forward-Looking StatementsThis press release contains certain statements that are forward-looking within the meaning of Section 27A of the Securities Act of 1933, as amended, and Section 21E of the Securities Exchange Act of 1934, as amended, and that involve risks and uncertainties. These statements include statements about the Company exploring all strategic options, including the sale of some or all of its assets or sale of the Company. We have attempted to identify forward-looking statements by such terminology as may, will, believe, estimate, expect, and similar expressions (as well as other words or expressions referencing future events, conditions or circumstances), which constitute and are intended to identify forward-looking statements. Actual results may differ materially from those indicated by such forward-looking statements as a result of various important factors, numerous risks and uncertainties, including but not limited to the potential impacts of the COVID-19 pandemic on our business, operations, and financial condition, the outcome of the strategic review, continued interest in our rare disease portfolio, our ability to enroll patients in clinical trials, the outcome of any future meetings with the U.S. Food and Drug Administration or other regulatory agencies, the impact of competition, the ability to secure licenses for any technology that may be necessary to commercialize our products, the ability to achieve or obtain necessary regulatory approvals, the impact of changes in the financial markets and global economic conditions, risks associated with data analysis and reporting, and other risks disclosed in the Companys most recent Annual Report on Form 10-K and subsequent quarterly reports on Form 10-Q and other periodic reports filed with the Securities and Exchange Commission. The Company undertakes no obligation to revise the forward-looking statements or to update them to reflect events or circumstances occurring after the date of this press release, whether as a result of new information, future developments or otherwise, except as required by the federal securities laws.

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Abeona Therapeutics Announces Acceptance of Late-Breaker Abstracts Highlighting New Clinical Data for Novel AAV-based Gene Therapies in MPS IIIA and...

DUBLIN--(BUSINESS WIRE)--The "Lab Automation in Genomics Market - Growth, Trends, and Forecast (2020 - 2025)" report has been added to ResearchAndMarkets.com's offering.

The Global Lab Automation in the Genomics market is expected to register a CAGR approximated at around 7.5% during the period of 2020-2025.

With the technological advancements and increasing computational capacities, there has been significant improvement in knowledge of genome sequencing in terms of data analytics advances that show unknown correlations, hidden patterns, and other insights, especially when it comes to testing data sets a large scale.

Moreover, novel advances in medicine are being made at a rapidly increasing pace, largely due to recent developments in genome analysis. DNA sequence analysis provides a clearer understanding of how genetic variation leads to disease and will ultimately lead to new cures. Also, laboratory automation has proved to make room for great flexibility, higher throughputs, and affordable solutions. It offers faster handling and process can be expedited without the worry for lack of reliability and precision. Genotyping and DNA sequencing have been very affordable, due to which the rate of growth is robust.

Advances in DNA sequencing technology have made it cost-effective and more accessible than before, which has led to a flurry of genetic testing start-ups. To bring the price down, companies, such as Color Genomics and Counsyl, have built their sequencing labs and analytics software from scratch, using robots and machine learning to optimize operations.

Various genomics applications that can integrate automation in their processes include nucleic acid isolation, RNAi screening, CRISPR analysis, PCR, and gene expression analysis. Players/Vendors in the laboratory automation are designing tools that cater to these application needs. For instance, Explorer G3 integrated workstations are developed by PerkinElmer, an American company focused in the business areas of life science research, and industrial testing, to meet customers' automation needs for genomics applications.

Moreover, the companies' other kind of product launches through partnerships, which is increasing the market share of automation in the genomics. For instance, in January 2020, pentrons Labworks, Inc. and Swift Biosciences, Inc. announced the launch of the most affordable fully-automated workstation for next-generation sequencing (NGS) library preparation ever put on the market.

Since the beginning of the COVID-19 epidemic, labs have been converting their spaces and resources into COVID-19 testing facilities, leading to increased adoption of automation equipment. The labs at the University of Washington were the first ones to do this. The Broad Institute followed an announcement of the conversion of their clinical processing lab into a large-scale COVID-19 testing facility.

Key Market Trends

Automated Liquid Handlers to Witness High Growth

Cross contamination is a major problem in the genomics laboratory, which can be avoided by implementing automated systems to manage the reagents and reaction mixtures. It is believed that taking out human intervention helps in achieving more consistency.

North America Occupies the Largest Market Share

North America has been a pioneer in clinical research for years. This region is home to some major pharmaceutical companies, like Pfizer, Novartis, GlaxoSmithKline, J&J, and Novartis. The region also has the highest concentration of contract research organizations (CROs). Some of the significant CROs are Laboratory Corp. of America Holdings, IQVIA, Syneos Health, and Parexel International Corp.

Owing to the presence of all the major players in the industry and stringent FDA regulations, the market is very competitive in the region. To gain an advantage over competitors, the genomics research organizations in the region are increasingly adopting robotics and automation in labs.

Competitive Landscape

The lab automation in the genomics market is a competitive market, owing to the presence of many small and big players exporting products to many countries. The key strategies adopted by the major players are a technological advancement in the product, partnerships, and merger and acquisition.

Some of the major players in the market are Thermo Fisher Scientific Inc., Becton Dickinson, Siemens Healthineers AG, and Tecan Group Ltd, among others.

Key Topics Covered:

1 INTRODUCTION

1.1 Study Assumptions & Market Definition

1.2 Scope of the Study

2 RESEARCH METHODOLOGY

3 EXECUTIVE SUMMARY

4 MARKET DYNAMICS

4.1 Market Overview

4.2 Industry Attractiveness - Porter's Five Forces Analysis

4.3 Market Drivers

4.3.1 Growing Trend of Digital Transformation for Laboratories with IoT

4.3.2 Effective Management of the Huge Amount of Data Generated

4.4 Market Restraints

4.4.1 Expensive Initial Setup

4.5 Impact of COVID-19 on the Lab Automation Market

5 MARKET SEGMENTATION

5.1 By Equipment

5.1.1 Automated Liquid Handlers

5.1.2 Automated Plate Handlers

5.1.3 Robotic Arms

5.1.4 Automated Storage and Retrieval Systems (AS/RS)

5.1.5 Vision Systems

5.2 By Geography

5.2.1 North America

5.2.2 Europe

5.2.3 Asia-Pacific

5.2.4 Rest of the World

6 COMPETITIVE LANDSCAPE

6.1 Company Profiles

6.1.1 Thermo Fisher Scientific Inc.

6.1.2 Danaher Corporation / Beckman Coulter

6.1.3 Hudson Robotics, Inc.

6.1.4 Becton, Dickinson and Company

6.1.5 Synchron Lab Automation

6.1.6 Agilent Technologies Inc.

6.1.7 Siemens Healthineers AG

6.1.8 Tecan Group Ltd

6.1.9 Perkinelmer Inc.

6.1.10 Honeywell International

6.1.11 Eppendorf AG

7 INVESTMENT ANALYSIS

8 MARKET OPPORTUNITIES AND FUTURE TRENDS

For more information about this report visit https://www.researchandmarkets.com/r/nnxcio

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Lab Automation in Genomics Market - Global Growth, Trends, and Forecast 2020-2025 with Thermo Fisher Scientific, Becton Dickinson, Siemens...

PARIS--(BUSINESS WIRE)--Regulatory News:

GenSight Biologics (Paris:SIGHT) (Euronext: SIGHT, ISIN: FR0013183985, PEA-PME eligible), a biopharma company focused on developing and commercializing innovative gene therapies for retinal neurodegenerative diseases and central nervous system disorders, today announced that the journal Science Translational Medicine has published results from the REVERSE pivotal Phase III clinical trial of LUMEVOQ gene therapy in ND4 Leber Hereditary Optic Neuropathy (LHON) subjects along with key results from a non-human primate study investigating the contralateral effect of the gene therapy. The paper*, published in the December issue under the title Bilateral visual improvement with unilateral gene therapy injection for Leber hereditary optic neuropathy, is the first peer-reviewed article based on Phase III clinical trial data to document sustained and clinically meaningful bilateral improvement in visual outcomes from a unilateral injection of a gene therapy.

The findings from the REVERSE trial and the non-human primate study were key components of the data package submitted by GenSight Biologics in September 2020 to the European Medicines Agency when it applied for marketing authorization for LUMEVOQ as treatment for patients with visual loss due to LHON caused by a confirmed mutation in the ND4 mitochondrial gene. The agencys decision is expected in Q4 2021.

The treatment has been shown to be safe and the outcomes can be life changing, said Dr. Patrick Yu-Wai-Man, MD, PhD, lead author, REVERSE principal investigator and Senior Lecturer and Honorary Consultant Ophthalmologist at the University of Cambridge, Moorfields Eye Hospital, and the UCL Institute of Ophthalmology, London, United Kingdom.

Our study provides great hope for treating this blinding disease in young adults, said Dr. Jos-Alain Sahel, MD, co-corresponding author, co-founder of GenSight and Director of the Institut de la Vision (Sorbonne-Universit/Inserm/CNRS), Paris, France, where LUMEVOQs underlying mitochondrial targeting technology was developed. Our approach isnt just limited to vision restoration: other mitochondrial diseases could be treated using the same technology, added Dr. Sahel, who is also Chairman of the Department of Ophthalmology at Centre Hospitalier National dOphtalmologie des XV-XX, Paris, France, and Professor and Chairman of the Department of Ophthalmology at the University of Pittsburgh School of Medicine and UPMC (University of Pittsburgh Medical Center), USA.

REVERSE Trial outcomes

37 ND4 LHON subjects, who experienced onset of vision loss from 6 months to one year before enrollment, participated in the REVERSE trial. The results show a clinically meaningful improvement over baseline of +15 ETDRS letters (0.308 LogMAR) in the average best-corrected visual acuity (BCVA) of injected eyes of the 37 REVERSE patients 96 weeks after treatment. The patients other eye, which received a sham injection, experienced an average visual acuity gain over baseline of +13 letters equivalent (0.259 LogMAR). Against nadir, or the worst recorded BCVA, the gains were even more impressive, at +28.5 ETDRS letters for the LUMEVOQ-injected eyes and +24.5 ETDRS letters for sham-injected eyes.

81% of subjects showed a clinically relevant recovery (CRR) from the nadir in one or both eyes. CRR, a measure of treatment response established by an international consensus meeting on the management of LHON1, is defined as either an improvement from off-chart BCVA to on-chart, or an on-chart improvement BCVA of at least -0.2 LogMAR, or +10 ETDRS letters.

The improvement in quality of life metrics relative to baseline values taken before treatment, which were evaluated using the well-established National Eye Institute Visual Function Questionnaire-25 (NEI VFQ-25), was compelling and largely above the thresholds of clinical relevance. The composite NEI VFQ-25 score showed a mean improvement of 9.5 points, exceeding the clinically relevant threshold of +3.9-+4.3 points.2

Non-human primate study outcomes

The non-human primate study, launched to investigate the mechanism behind the unexpected improvement in the contralateral eyes visual function, was designed to mimic the REVERSE trial, with monkeys given a unilateral LUMEVOQ injection. The study demonstrated the transfer of viral vector DNA from the injected eye to the anterior segment, retina, and optic nerve of the noninjected eye. This result, the authors conclude, provides a plausible mechanistic explanation for the bilateral improvement in visual function after unilateral LUMEVOQ injection.

Other topics discussed

The paper also presents detailed safety data, which document the overall good safety profile of LUMEVOQ, with no viral vector biodissemination and mostly mild ocular adverse events that were controlled with local topical therapy. Additionally, the authors discuss other results from REVERSE, such as other responder analyses and visual outcomes, and place these in context against the natural history insights found in analyses of visual acuity in non-treated patients.

The paper can be obtained from http://www.sciencemag.org.

*About the paper:

Bilateral visual improvement with unilateral gene therapy injection for Leber hereditary optic neuropathy

Authors: Patrick Yu-Wai-Man1,2,3,4, Nancy J. Newman5, Valerio Carelli6,7, Mark L. Moster8, Valerie Biousse5, Alfredo A. Sadun9, Thomas Klopstock10,11,12, Catherine Vignal-Clermont13,14, Robert C. Sergott8, Gnther Rudolph15, Chiara La Morgia6,7, Rustum Karanjia9,16, Magali Taiel17, Laure Blouin17, Pierre Burguire17, Gerard Smits18, Caroline Chevalier17, Harvey Masonson18, Yordak Salermo18, Barrett Katz18, Serge Picaud19, David J. Calkins20, Jos-Alain Sahel14,19,21,22

Affiliations:

1 Cambridge Centre for Brain Repair and MRC Mitochondrial Biology Unit, Department of Clinical Neurosciences, University of Cambridge, Cambridge CB2 0PY, UK.2 Cambridge Eye Unit, Addenbrookes Hospital, Cambridge University Hospitals, Cambridge CB2 0QQ, UK.3 Moorfields Eye Hospital, London EC1V 2PD, UK.4 UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK.5 Departments of Ophthalmology, Neurology and Neurological Surgery, Emory University School of Medicine, Atlanta, GA 30322, USA.6 IRCCS Istituto delle Scienze Neurologiche di Bologna, UOC Clinica Neurologica, 40139 Bologna, Italy.7 Unit of Neurology, Department of Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, 40139 Bologna, Italy.8 Departments of Neurology and Ophthalmology, William H. Annesley, Jr. EyeBrain Center, Wills Eye Hospital and Thomas Jefferson University, Philadelphia, PA 19107, USA.9 Doheny Eye Institute and UCLA School of Medicine, Los Angeles, CA 90086, USA.10 Friedrich Baur Institute at the Department of Neurology, University Hospital, LMU Munich, 80336 Munich, Germany.11German Center for Neurodegenerative Diseases (DZNE), 80336 Munich, Germany.12 Munich Cluster for Systems Neurology (SyNergy), 80336 Munich, Germany.13 Department of Neuro-Ophthalmology and Emergencies, Rothschild Foundation Hospital, 75019 Paris, France.14 Centre Hospitalier National dOphtalmologie des Quinze Vingts, FOReSIGHT, INSERM-DGOS CIC 1423, 75012 Paris, France. 15Department of Ophthalmology, University Hospital, LMU Munich, 80336 Munich, Germany.16 Ottawa Hospital Research Institute and University of Ottawa Eye Institute, Ottawa, Ontario K1H 8L6, Canada.17 GenSight Biologics, 75012 Paris, France.18 GenSight Biologics, New York, NY 10016, USA.19 Sorbonne Universit, INSERM, CNRS, Institut de la Vision, 75012 Paris, France.20 The Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, TN 37232, USA.21 Fondation Ophtalmologique A. de Rothschild, 25-29 Rue Manin, 75019 Paris, France.22 Department of Ophthalmology, The University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.

Notes:

1 V. Carelli, M. Carbonell, I. F. de Coo, A. Kawasaki, T. Klopstock, W. A. Lagrze, C. La Morgia, N. J. Newman, C. Orssaud, J. W. R. Pott, A. A. Sadun, J. van Everdingen, C. Vignal-Clermont, M. Votruba, P. Yu-Wai-Man, P. Barboni, International consensus statement on the clinical and therapeutic management of Leber hereditary optic neuropathy. J. Neuroophthalmol. 37, 371381 (2017).2 I. J. Suer, G. T. Kokame, E. Yu, J. Ward, C. Dolan, N. M. Bressler, Responsiveness of NEI VFQ-25 to changes in visual acuity in neovascular AMD: Validation studies from two phase 3 clinical trials. Invest. Ophthalmol. Vis. Sci. 50, 36293635 (2009).

About GenSight Biologics

GenSight Biologics S.A. is a clinical-stage biopharma company focused on developing and commercializing innovative gene therapies for retinal neurodegenerative diseases and central nervous system disorders. GenSight Biologics pipeline leverages two core technology platforms, the Mitochondrial Targeting Sequence (MTS) and optogenetics, to help preserve or restore vision in patients suffering from blinding retinal diseases. GenSight Biologics lead product candidate, LUMEVOQ (GS010; lenadogene nolparvovec), has been submitted for marketing approval in Europe for the treatment of Leber Hereditary Optic Neuropathy (LHON), a rare mitochondrial disease affecting primarily teens and young adults that leads to irreversible blindness. Using its gene therapy-based approach, GenSight Biologics product candidates are designed to be administered in a single treatment to each eye by intravitreal injection to offer patients a sustainable functional visual recovery.

About Leber Hereditary Optic Neuropathy (LHON)

Leber Hereditary Optic Neuropathy (LHON) is a rare maternally inherited mitochondrial genetic disease, characterized by the degeneration of retinal ganglion cells that results in brutal and irreversible vision loss that can lead to legal blindness, and mainly affects adolescents and young adults. LHON is associated with painless, sudden loss of central vision in the 1st eye, with the 2nd eye sequentially impaired. It is a symmetric disease with poor functional visual recovery. 97% of patients have bilateral involvement at less than one year of onset of vision loss, and in 25% of cases, vision loss occurs in both eyes simultaneously. The estimated incidence of LHON is approximately 800-1,200 new patients who lose their sight every year in the United States and the European Union.

About LUMEVOQ (GS010)

LUMEVOQ (GS010) targets Leber Hereditary Optic Neuropathy (LHON) by leveraging a mitochondrial targeting sequence (MTS) proprietary technology platform, arising from research conducted at the Institut de la Vision in Paris, which, when associated with the gene of interest, allows the platform to specifically address defects inside the mitochondria using an AAV vector (Adeno-Associated Virus). The gene of interest is transferred into the cell to be expressed and produces the functional protein, which will then be shuttled to the mitochondria through specific nucleotidic sequences in order to restore the missing or deficient mitochondrial function. LUMEVOQ was accepted as the invented name for GS010 (lenadogene nolparvovec) by the European Medicines Agency (EMA) in October 2018.

About RESCUE and REVERSE

RESCUE and REVERSE are two separate randomized, double-masked, sham-controlled Phase III trials designed to evaluate the efficacy of a single intravitreal injection of GS010 (rAAV2/2-ND4) in subjects affected by LHON due to the G11778A mutation in the mitochondrial ND4 gene.

The primary endpoint measured the difference in efficacy of GS010 in treated eyes compared to sham-treated eyes based on BestCorrected Visual Acuity (BCVA), as measured with the ETDRS at 48 weeks post-injection. The patients LogMAR (Logarithm of the Minimal Angle of Resolution) scores, which are derived from the number of letters patients read on the ETDRS chart, was used for statistical purposes. Both trials were adequately powered to evaluate a clinically relevant difference of at least 15 ETDRS letters between treated and untreated eyes adjusted to baseline.

The secondary endpoints involved the application of the primary analysis to bestseeing eyes that received GS010 compared to those receiving sham, and to worseseeing eyes that received GS010 compared to those that received sham. Additionally, a categorical evaluation with a responder analysis was evaluated, including the proportion of patients who maintain vision (< ETDRS 15L loss), the proportion of patients who gain 15 ETDRS letters from baseline and the proportion of patients with Snellen acuity of >20/200. Complementary vision metrics included automated visual fields, optical coherence tomography, and color and contrast sensitivity, in addition to quality of life scales, biodissemination and the time course of immune response. Readouts for these endpoints were at 48, 72 and 96 weeks after injection.

The trials were conducted in parallel, in 37 subjects for REVERSE and 39 subjects for RESCUE, in 7 centers across the United States, the UK, France, Germany and Italy. Week 96 results were reported in 2019 for both trials, after which patients were invited to a long-term follow-up study that will last for three years.

ClinicalTrials.gov Identifiers:REVERSE: NCT02652780RESCUE: NCT02652767

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GenSight Biologics Announces Publication of Results from LUMEVOQ REVERSE Pivotal Phase III Trial and Non-Human Primate Study in Science Translational...

Round was led by Sofinnova Investments with participation from Abingworth, Lightstone Ventures and all existing investors

Company expands board of directors and plans to build out team

DURHAM, N.C. and BOSTON, Dec. 16, 2020 (GLOBE NEWSWIRE) -- Atsena Therapeutics, a clinical-stage gene therapy company focused on bringing the life-changing power of genetic medicine to reverse or prevent blindness, today announced it has closed an oversubscribed $55 million Series A financing led by Sofinnova Investments with participation from additional new investors Abingworth and Lightstone Ventures. Founding investors Hatteras Venture Partners and the Foundation Fighting Blindness RD Fund, along with existing investors Osage University Partners, University of Florida, and Manning Family Foundation, also participated in the round. Sarah Bhagat, PhD, Partner at Sofinnova, Jackie Grant, PhD, Principal at Abingworth, and Jason Lettmann, General Partner at Lightstone, will join Atsenas board of directors.

Proceeds will be used to advance Atsenas ongoing Phase I/II clinical trial evaluating a gene therapy for patients with GUCY2D-associated Leber congenital amaurosis (LCA1), one of the most common causes of blindness in children, as well as complete manufacturing development for Phase 3. In addition, the funds will enable Atsena to expand its team to support the research and development of novel gene therapies, including the progression of two existing preclinical programs in inherited retinal diseases toward the clinic and advancement of the companys innovative adeno-associated virus (AAV) technology platform.

We are grateful for the support of our new and existing investors and are encouraged by their enthusiasm for the potential of our technology to overcome the unique hurdles of inherited retinal diseases to prevent or reverse blindness, said Patrick Ritschel, MBA, Chief Executive Officer of Atsena. The Series A financing provides financial runway to reach the key inflection point of reading out efficacy data from our LCA1 clinical trial. While we continue expeditiously advancing this trial and our preclinical programs, we are excited to accelerate our growth as a leading ophthalmic gene therapy company.

The Phase I/II LCA1 clinical trial is currently enrolling patients in the second dosing cohort. Atsena exclusively licensed the rights to the gene therapy from Sanofi, which originally licensed it from University of Florida. LCA is the most common cause of blindness in children. LCA1 is caused by mutations in the GUCY2D gene and results in early and severe vision impairment or blindness. GUCY2D-LCA1 is one of the most common forms of LCA, affecting roughly 20 percent of patients who live with this inherited retinal disease.

We believe Atsenas foundation in ocular gene therapy and potentially game-changing novel AAV vectors position the company to become a partner of choice, said Dr. Bhagat. Sofinnova is delighted to support Atsena and we look forward to helping the team further its mission to develop life-changing gene therapies for patients with inherited retinal diseases.

About Atsena TherapeuticsAtsena Therapeutics is a clinical-stage gene therapy company developing novel treatments for inherited forms of blindness. The companys ongoing Phase I/II clinical trial is evaluating a potential therapy for one of the most common causes of blindness in children. Its additional pipeline of leading preclinical assets is powered by an adeno-associated virus (AAV) technology platform tailored to overcome significant hurdles presented by inherited retinal disease, and its unique approach is guided by the specific needs of each patient condition to optimize treatment. Founded by ocular gene therapy pioneers Dr. Shannon Boye and Sanford Boye, Atsena has a licensing, research and manufacturing collaboration with the University of Florida and has offices in Boston, MA and North Carolinas Research Triangle, environments rich in gene therapy expertise. For more information, please visit atsenatx.com.

About Sofinnova InvestmentsSince our founding in 1974, Sofinnova has been active in life science investing. We are a clinical-stage biopharmaceutical investment firm with approximately $2.3B in assets under management and committed capital. We invest in both private and public equity of therapeutics-focused companies. Our goal is to actively partner with entrepreneurs in both the U.S. and Europe, across all stages of company formation. From drug development and navigating the regulatory process to company building and IPO, we strive to be collaborative, meaningful board members, and excellent partners at every level. We seek to build world class companies that aspire to dramatically improve the current state of medical care today and ultimately, the lives of patients. Sofinnova has expertise investing in gene therapy companies, including investments in Spark, which developed the first approved gene therapy, Akouos, and Audentes, and Xylocor. For more information, please visit http://www.sofinnova.com.

About Abingworth Abingworth is a leading transatlantic life sciences investment firm. Abingworth helps transform cutting-edge science into novel medicines by providing capital and expertise to top caliber management teams building world-class companies. Since 1973, Abingworth has invested in approximately 168 life science companies, leading to more than 44 M&A/exits and close to 70 IPOs. Our therapeutic focused investments fall into 3 categories: seed and early-stage, development stage, and clinical co-development. Abingworth supports its portfolio companies with a team of experienced professionals at offices in London, Menlo Park (California) and Boston. For more information, visit abingworth.com.

About Lightstone VenturesLightstone Ventures is a leading venture capital firm investing in therapeutic-oriented companies across the life science spectrum, from breakthrough medical devices to novel drugs and biopharmaceuticals. Founded in 2012, Lightstone has been part of many successful new ventures from inception through commercialization and plays a critical role guiding and building successful healthcare companies. With a proven strategy and global footprint, the Lightstone team has been involved in several of the largest venture-backed life science exits over the last decade including: ALX Oncology, Acceleron, Ardian, Calithera, Claret Medical, Disarm, MicroVention, Nimbus, Plexxikon, Portola, Promedior, Proteolix, Ra Pharma, Tizona, Twelve and Zeltiq. For more information, visithttps://www.lightstonevc.com.

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Atsena Therapeutics Raises $55 Million Series A Financing to Advance LCA1 Gene Therapy Clinical Program, Two Preclinical Assets, and Novel Capsid...

J.P. Morgan Securities LLC, Cowen and Company, LLC, and Credit Suisse Securities (USA) LLC are acting as joint book-running managers in the offering. Canaccord Genuity LLC and Cantor Fitzgerald & Co. are acting as passive bookrunners in the offering.

Stoke intends to use the net proceeds from the proposed offering, together with its existing cash and cash equivalents, to fund research, clinical and process development and manufacturing of its product candidates, including late stage development of STK-001, clinical development of its next target for the treatment of Autosomal Dominant Optic Atrophy, developing additional product candidates, working capital, capital expenditures and other general corporate purposes.

The shares are being offered by Stoke pursuant to a registration statement on Form S-3 previously filed and declared effective by the Securities and Exchange Commission (the SEC). A preliminary prospectus supplement and accompanying prospectus relating to this offering have been filed with the SEC. Copies of the preliminary prospectus supplement and the accompanying prospectus relating to this offering, and when available, the final prospectus supplement, may be obtained from: J.P. Morgan Securities LLC, c/o Broadridge Financial Services, Attention: Prospectus Department, 1155 Long Island Avenue, Edgewood, New York 11717, or by telephone: (866) 803-9204, or by emailing prospectus-eq_fi@jpmchase.com; from Cowen and Company, LLC c/o Broadridge Financial Solutions, Attention: Prospectus Department, 1155 Long Island Avenue, Edgewood, New York 11717, or by telephone: (833) 297-2926, or by emailing PostSaleManualRequests@broadridge.com; or from Credit Suisse Securities (USA) LLC, Attention: Prospectus Department, 6933 Louis Stephens Drive, Morrisville, NC 27560, by telephone at (800) 221-1037, or by email at usa.prospectus@credit-suisse.com. Electronic copies of the preliminary prospectus supplement and accompanying prospectus will also be available on the website of the SEC at http://www.sec.gov.

This press release shall not constitute an offer to sell or the solicitation of an offer to buy any securities of Stoke, nor shall there be any sale of these securities in any state or jurisdiction in which such offer, solicitation or sale would be unlawful prior to registration or qualification under the securities laws of any such state or jurisdiction.

About Stoke Therapeutics

Stoke Therapeutics (Nasdaq: STOK) is a biotechnology company pioneering a new way to treat the underlying causes of severe genetic diseases by precisely upregulating protein expression to restore target proteins to near normal levels. Stoke aims to develop the first precision medicine platform to target the underlying cause of a broad spectrum of genetic diseases in which the patient has one healthy copy of a gene and one mutated copy that fails to produce a protein essential to health. These diseases, in which loss of approximately 50% of normal protein expression causes disease, are called autosomal dominant haploinsufficiencies. Stoke is headquartered in Bedford, Massachusetts with offices in Cambridge, Massachusetts.

Forward-Looking Statements

This press release contains forward-looking statements within the meaning of the safe harbor provisions of the Private Securities Litigation Reform Act of 1995. Forward-looking statements can be identified by words such as: anticipate, intend, plan, goal, seek, believe, project, estimate, expect, strategy, future, likely, may, should, will and similar references to future periods. Examples of forward-looking statements include, among others, statements the Company makes regarding its expectation of market conditions and the satisfaction of customary closing conditions related to the offering, its ability to complete the offering and expected use of proceeds, Stokes plan to develop its precision medicine platform, anticipated preclinical and clinical development activities, potential benefits of Stokes product candidates and potential market opportunities for Stokes product candidates. All statements other than statements of historical fact are statements that could be deemed forward-looking statements. Although Stoke believes that the expectations reflected in such forward-looking statements are reasonable, Stoke cannot guarantee future events, results, actions, levels of activity, performance or achievements, and the timing and results of biotechnology development and potential regulatory approval is inherently uncertain. Forward-looking statements are subject to risks and uncertainties that may cause the Companys actual activities or results to differ significantly from those expressed in any forward-looking statement, including risks and uncertainties related to the impact of the COVID-19 pandemic on the Companys business, clinical trial sites, supply chain and manufacturing facilities, market conditions, the satisfaction of customary closing conditions related to the proposed offering, as well as other risks and uncertainties described under the heading Risk Factors in documents Stoke files from time to time with the SEC. These forward-looking statements speak only as of the date hereof and Stoke specifically disclaims any obligation to update these forward-looking statements or reasons why actual results might differ, whether as a result of new information, future events or otherwise, except as required by law.

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Stoke Therapeutics Announces Pricing of $97.5 Million Public Offering - BioSpace