Gene Therapy advance online publication 15January2015; doi: 10.1038/gt.2014.124

First-generation (E1 deleted) adenovirus vectors (FG AdVs), which lack the E1 and E3 regions, are popularly used in basic studies to elucidate gene functions, and have been employed for gene therapy.1, 2, 3, 4 Because the DNA fragments of up to about 7 kilobases (kb) in total can be inserted into the AdV genome, the AdVs are frequently used to produce two proteins simultaneously from two independent transgenes expressing both the target gene and the reporter gene, for example. In the studies using the cultured cells and in the animal experiments, the GFP and luciferase are used as the reporters. Recently, positron emission tomography has clinically been used in patients for diagnoses and in experimental animal models. Therefore, the AdVs containing both the therapeutic gene and the positron emission tomography reporter gene would be valuable in the gene therapy fields, because the therapeutic effects, the vector duration and distribution can simultaneously be monitored.5, 6, 7, 8 Probably one would wish for high-titer AdVs with the highest expression for the therapeutic gene and with the second highest for the reporter gene not causing any trouble, if the insertion sites and orientations in the AdV genome can be chosen. However, the titers and the expression levels of the AdVs may considerably be influenced by the sites and orientations of the transgenes. Such information may be very valuable for construction of the best vector, especially in the vector containing both the therapeutic gene and the reporter gene.

The simultaneous expression of two genes could be achieved by inserting the two genes into the E1 site under the control of a single prompter using the internal ribosomal entry sites or using porcine teschovirus-1 2A.9, 10 In the former approach, the expression of the second gene might be influenced by the sequences between internal ribosomal entry sites and its initiation codon, and in the latter, the manipulation is necessary to remove the stop codon of the first gene and to adjust the frames of the two genes. When two genes driven by the independent promoters are inserted into the E1 site, they might interfere with each other. However, when two independent expression units are inserted in different sites in the AdV genome, no interference occurs. Moreover, the advantage of this approach is that the main target gene can easily be changed using the AdV cassette that already contains the reporter gene.

There are three insertion sites and two orientations: a transgene can be inserted into the AdV genome by substitution of the E1 or E3 gene and by simple insertion at a position upstream of the E4 gene. Therefore, there are six different possible sites/orientations for any given transgene. Moreover, not only the potent promoters such as EF1 but also tissue-specific promoters such as -fetoprotein (AFP) can also be employed. Although the studies examining which sites/orientations are superior to others are practically important, they have been very limited11, 12 and systematic analyses have not been reported so far.

As it is known that the expression level of a transgene varies considerably depending on the site in the cell chromosome of the human genome, the phenomenon is called the position effect.13, 14 Although CG-methylation in the cell chromosome is clearly one reason, it is not observed in the AdV genome. Therefore, it would be of interest to examine whether the position effect might also be observed similarly in the AdV genome for the potent promoter and for the tissue-specific promoter.

FG AdVs retain almost all viral genes. They are normally not expressed in the target cells, because E1A protein, the essential transactivator for expression of all other viral genes, is not present. However, there is one report of splicing of aberrant mRNAs from the inserted foreign genes to a viral gene.15 In this case, the aberrant mRNAs are transcribed by strong foreign promoters and produce transgene-viral gene fusion proteins, which elicit strong immune responses. However, it is not known whether the production of the aberrant gene product between the inserted transgene and viral gene is rare or not.

In this study, we examined the AdV titers and expression levels of an identical transgene inserted at the E1, E3 and E4 sites. We used three transgenes, namely, GFP, LacZ and Cre, and two promoters, namely, the potent EF1 promoter and the cancer-specific AFP promoter, and attempted to construct AdVs using all combinations, that is, 18 AdVs, and succeeded in constructing 17 of them. We found that insertion at the E1 and E4 sites yielded mostly high titers, whereas the one at the E3 yielded variable titers. Surprisingly, four aberrantly spliced mRNAs between the transgenes and viral genes were found in the vector obtained by insertion at the E3 site, which was probably the reason for the very low titers. As for the expression levels, clear differences were observed among the vectors obtained with insertion at the E1, E3 and E4 sites despite using the identical transgene, indicating that the position effect was certainly present for the AdV genome and that aberrant splicing may, at least in part, explain this effect. We also propose a strategy to avoid generation of the aberrantly spliced mRNAs.

We first examined whether the vector titers were influenced by the site/orientations of the transgenes containing a potent EF1 promoter. Towards this end, we attempted to construct six GFP-expressing (EF-GFP) and six LacZ-expressing (EF-LacZ) vectors in all possible combinations, that is, the E1, E3 and E4 insertion sites and the two orientations ( Figure 1), and measured the vector titers (Figure 2a) (hereinafter, the vectors will be designated as per the following; the vectors containing the GFP gene and LacZ gene at the E1 insertion site and in the left orientation shall be denoted as G-E1L and Z-E1L vectors, respectively). Among the GFP-expressing vectors, high titers were obtained for G-E1L, G-E3L, G-E4L and G-E4R vectors (Figure 2a, bars 1, 3, 5 and 6), while the titer for the G-E1R vector was lower (bar 2). Notably, the G-E3R vector, that is, vector with the GFP transgene inserted in the E3 site in the rightward orientation, could not be obtained despite three independent attempts (bar 4, denote ). Therefore, although exactly the same EF1-GFP expression unit was inserted in these vectors, the sites and orientations exerted considerable influence on the vector titers and even determined whether the vector was available or not. Similar results were obtained for vectors expressing LacZ: the titers of the Z-E1L, Z-E4L and Z-E4R vectors (bars 7, 11 and 12) were high, and that of the Z-E1R vector was also low (bar 8). However, the results of insertion at the E3 site differed for GFP and LacZ. The titer ratio of Z-E3L was significantly lower than that of G-E3L (compare bars 3 and 9, described later), and the Z-E3R vector was available, although its titer was extremely low (bar 10). Therefore, the GFP gene and LacZ gene themselves influenced the vector titers.

The FG AdV structures of six different site/orientations in all possible combinations. The box containing pro, gene and pA represents the expression unit and the arrows show the orientation of transcription. pro, EF1 and AFP promoter; gene, GFP, LacZ and Cre; pA, rabbit -globin polyadenylation signal. For example, the vector containing the transgene at the E1 insertion site and in the left orientation is denoted as E1L.

Titers of the virus vectors containing identical expression units. (a) Virus titers of the AdVs containing the EF1 promoter. The AdV genomes transduced into the HuH-7 cells were measured 3 days post infection. The virus titers were calculated relative to the copy numbers of the AdVs.16 The titer of the E1L vector was set as 1; G-E1L, 8.3 108 relative virus titer (rVT)/ml, L-E1L, 5.0 109 rVT/ml. indicates that G-E3R could not be obtained. (b) The titers of the virus vector containing Cre gene driven by the AFP promoter. E1L vector was used as the control. *P<0.05, **P<0.01.

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Juvenile Arthritis report gene therapy approach
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An exhaustive hereditary analysis of a large Louisiana family with vision issues has uncovered a new gene tied to an incurable eye disorder called retinitis pigmentosa, according to an examination led by scientists at The University of Texas Health Science Center at Houston (UTHealth). It is a family of eye diseases that affects more than 200,000 in the United States and millions worldwide

The retina converts images into electrical signals that can be processed by the brain. It acts much like the film in a camera. Retinitis pigmentosa damages this film (the retina) and its early symptoms include decreased night vision and peripheral vision. Once it starts, the loss of vision is relentlessly progressive, often ending in blindness.

In the journal Investigative Ophthalmology & Visual Science, UTHealth's Stephen P. Daiger, Ph.D., and his colleagues report their discovery of a new gene tied to retinitis pigmentosa, which brings the total of genes associated with this sight-threatening disease to more than 60. The gene is called hexokinase 1 (HK1).

This information is important because it helps affected families cope with the disorder, helps explain the biologic basis of these diseases and suggests targets for drug treatments and gene therapy, said Daiger, the report's senior author and holder of the Thomas Stull Matney Ph.D. Endowed Professorship in Environmental and Genetic Sciences at UTHealth School of Public Health.

"The challenge now is to block the activity of these mutations and clinical trials are underway to do just that," he said.

"Dr. Daiger is trying to make a breakthrough in potentially blinding diseases with no known treatments," said Richard S. Ruiz, M.D., professor of ophthalmology and holder of the John S. Dunn Distinguished University Chair in Ophthalmology at UTHealth. "Right now, we address the symptoms of the disease and help patients make the most of their existing vision."

For approximately three decades, Daiger, a member of the Human Genetics Center at the UTHealth School of Public Health, has been following the progress of hundreds of families across the country with retinitis pigmentosa. "We've found the cause of disease in 80 percent of the families we have studied," Daiger said. "Our goal is to find the cause in the remaining 20 percent."

Equipped with the genetic profiles of family members, Daiger's team has identified differences in the genetic makeup of those with the disease. The researchers also use family histories and DNA tests to glean information about the condition's hereditary nature.

There are different types of retinitis pigmentosa and Daiger's laboratory is focused on the autosomal dominant type. This means that only one parent needs the mutation in order to pass the disease to a child. This type accounts for about a third of all cases and many of its disease-causing genes have been discovered, several by Daiger's research group.

"The story of the HK1 mutation is itself interesting. What we found is a mutation present in families from Louisiana, Canada and Sicily. Our evidence suggests the mutation arose in a common ancestor who lived centuries ago," Daiger said. "The mutation spread in Europe and North America, and may be common among Acadians in Louisiana. This is called a founder mutation."

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Worlds First Gene Therapy Trial To Cure Deafness - Nov 2014
DENVER - A local man is a making medical history. Rob Gerk is the first person in the world to receive gene therapy to restore his hearing. "It was very inte...

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Worlds First Gene Therapy Trial To Cure Deafness - Nov 2014 - Video

Gene Therapy (2015) 22, 2028; doi:10.1038/gt.2014.101; published online 6 November 2014

K LJackson1, R DDayton1, E AOrchard2, SJu3, DRinge4, G APetsko4,5, L EMaquat6,7 and R LKlein1

Amyotrophic lateral sclerosis (ALS) is a deadly neurodegenerative disease involving progressive paralysis. There are no highly efficacious strategies to treat ALS despite great effort by doctors and scientists. Successful treatments in mouse models, most of which are based on rare familial mutations in the ALS gene SOD1, have so far had little impact on modifying the disease in humans. Novel models based on transactive response DNA-binding protein 43kDa (TDP-43) may offer a more predictive test system given that the vast majority of ALS cases harbor TDP-43 pathology in their neurons and glia.1, 2, 3 Abnormal TDP-43 aggregates are also prominent in the class of diseases known as frontotemporal lobar degeneration (FTLD-TDP).4 TDP-43 is an RNA-binding protein that is normally found predominantly in the nucleus. In FTLD-TDP and the majority of ALS, abnormal TDP-43 accumulation occurs in the cytoplasm in the form of hyperphosphorylated and ubiquitinated pathological protein aggregates, and thus serves as a post-mortem diagnostic marker. 1, 2, 3, 4 One of the ways by which TDP-43 has been studied in animals is by gene delivery, which has proven to be sufficiently reproducible to allow the discrimination of genotypephenotype differences among TDP-43 isoforms in our previous work.5, 6, 7 This reproducibility and the ability to experimentally control the onset and severity of the disease state offer advantages for modeling, given that TDP-43 overexpression is highly toxic to cells.8 Here we use TDP-43 gene transfer to induce motor paralysis in rats to study limb symptomatology that is germane to ALS as a platform for gene therapy. Overexpression of TDP-43 causes progressive paresis to paralysis of the limbs in a highly reproducible manner,6, 7 offering an assay for therapeutic efficacy such as gene therapy. Gene therapy is worth considering for this disease given that ALS is fatal and irreversible. In this report, recombinant TDP-43 expression was titrated to a low level for a partial disease state retaining restorative capacity.

Refinement of TDP-43 animal models continues to be an important goal in the field.9 Reports of experimental treatments that slow or block TDP-43-mediated toxicity are beginning to emerge, either by genetic or pharmacological interventions in several TDP-43 models.10, 11, 12, 13, 14 Here we report behavioral outcomes from testing an empirically chosen therapeutic target, cDNA coding for human upframeshift protein 1 (UPF1), in a rat model of ALS-like paralysis based on TDP-43.

UPF1 is best known for its role in nonsense-mediated mRNA decay (NMD), a surveillance mechanism that degrades mRNAs containing a premature termination codon, which can be generated, for example, through alternative splicing. NMD prevents the production of truncated proteins that could harm the cell. NMD is also involved in the regulation of the expression of ~10% of normal physiologic transcripts in the cell, and is essential in mice.15, 16, 17 We pursued the possibility that UPF1 could ameliorate ALS-like symptoms based on the work carried out in yeast and neuronal cultures.18, 19 In a genetic screen of several thousands of proteins, a yeast homolog of hUPF1, and then the human gene itself, was found to prevent FUS- and TDP-43-mediated toxicity in yeast, 19 Ju et al., unpublished. Barmada et al.18 have advanced this approach, demonstrating that UPF1 protects primary neuronal cultures from TDP-43 cytotoxicity, possibly by upregulating NMD, as inhibitors of NMD blocked the protective effect. 18 The fact that expressing UPF1 blocks the toxic actions of TDP-43 in yeast cells and cultured neurons is consistent with the hypothesis that TDP-43-induced toxicity involves inhibition of UPF1 function, because TDP-43 toxicity can be suppressed by adding back UPF1 to restore NMD.

The main purpose of this study was to evaluate the expression of human mycUPF1 (i.e. human UPF1 with an N-terminal myc epitope tag) as a protection against TDP-43-induced limb paralysis in rats. MycUPF1 was tested in parallel with several different types of control treatments, all confirming that mycUPF1 elicits a specific therapeutic effect. We also evaluated whether the expression of recombinant TDP-43 or mycUPF1 would affect either each others recombinant gene expression or the expression of endogenous rat TDP-43 or UPF1. The data demonstrate that augmenting the cellular abundance of UPF1 provides a useful means of abrogating the devastating paralysis induced by TDP-43 overexpression.

Exogenous TDP-43 and green fluorescent protein (GFP) expression levels were purposefully set relatively low compared with the previous studies to test a rat model with a partial lesion and restorative capacity. This titration was advantageous to observe a therapeutic effect, but the low expression levels rendered detection of the transgene products inefficient. Nevertheless, previous work demonstrated that intravenous adenoassociated virus vector (AAV9) TDP-43 gene transfer specifically induces hindlimb paralysis even when the resulting level of exogenous TDP-43 is only faintly detectable.6 We chose the intravenous AAV9 method because it produces widespread central nervous system (CNS) expression, leading to marked expression in spinal motor neurons, dorsal root ganglia (DRG) neurons and cerebellar Purkinje neurons,6, 20 with only a small fold overexpression of the encoded protein, for example, less than twofold overexpression relative to the corresponding endogenous protein as estimated in the spinal cord in Dayton et al.7

For studying the effect of mycUPF1 expression, we harvested DRG neurons because this tissue provides a relatively high percentage of transduced cells in the nervous system, allowing for detection of transgene product. By comparison, the spinal cord and cerebellar samples include a greater percentage of non-transduced cells. We used antibodies for total TDP-43 or total UPF1 that detect both the endogenous rat plus exogenous human TDP-43 or UPF1. In DRG, the increase in total TDP-43 expression in AAV9 TDP-43/Empty vs uninjected animals was 2.4-fold (t-test, P<0.02, N=3 per group), whereas for total UPF1, we estimated the increase to be 1.6-fold in AAV9 mycUPF1 vs uninjected subjects ( Figure 1). The fold increases were relatively lower in the spinal cord (Figure 1) and cerebellum (not shown), as expected: the estimated ratio in the spinal cord and cerebellum was 1.4- and 1.2-fold for AAV9 TDP-43/Empty vs uninjected subjects and 1.1- and 1.1-fold for AAV9 mycUPF1 vs uninjected subjects (N=3 per group). Although fold overexpression levels were small, recombinant mycUPF1 could be specifically visualized using myc antibody, which detected recombinant mycUPF1 only in subjects receiving AAV9 mycUPF1 only or AAV9 TDP-43/mycUPF1 ( Figure 2).

Overexpression of TDP-43 or UPF1 in the rat CNS. Protein from dissected DRG and lumbar spinal cord was analyzed by western blotting 12 weeks after intravenous injection of AAV9 expression vectors. Three animals are shown for each condition. The level of total TDP-43 (endogenous rat plus recombinant human TDP-43) was significantly increased in the DRG of the AAV9 TDP-43/Empty group compared with uninjected subjects (t-test, P<0.02, N=3), but less so in the spinal cord or cerebellum (not shown). The expression level of human mycUPF1 compared with endogenous rat UPF1 was relatively small in all the three regions. The bands were normalized to GAPDH. See Results for details.

Selective detection of only recombinant human TDP-43 or mycUPF1. (a) A human-specific TDP-43 antibody detected exogenous human but not endogenous rat TDP-43 in DRGs. The level of TDP-43 expression was indistinguishable with or without mycUPF1 coexpression. (b) The level of exogenous mycUPF1 was detected with a myc antibody and only observed in rats that received AAV9 mycUPF1. In contrast to (a), MycUPF1 expression levels were reduced when AAV9 TDP-43 was coexpressed (t-test, P<0.05, N=3 for DRG). The bands were normalized to GAPDH.

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Gene therapy for cancer and other diseases
The recent two decades have seen dramatic progress in gene therapy, both scientifically and socially. We are now able to treat several single gene disorders,...

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4 Gene Therapy

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Gene Therapy To Restore Hearing Loss - Nov 2014
KANSAS CITY, Kan. Restoring hearing with a drug is the goal of a metro researcher. Now he #39;s begun the first study in humans of a gene therapy that could he...

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Gene Therapy To Restore Hearing Loss - Nov 2014 - Video

HOPE, HYPE REALITY: SCIENTIFIC RATIONALE FOR GENOME EDITING
Why we consider gene therapy to be a viable means to control or cure HIV.

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HOPE, HYPE & REALITY: SCIENTIFIC RATIONALE FOR GENOME EDITING - Video

Gene Therapy (2015) 22, 8795; doi:10.1038/gt.2014.85; published online 18 September 2014

Autosomal dominant familial hypercholesterolemia (FH) is caused by mutations in the low-density lipoprotein receptor (LDLR).1 Homozygous FH patients present with massively elevated LDL cholesterol (LDL-C) and cardiovascular disease. They have severe atherosclerosis and die of ischemic heart disease usually in their third decade of life. The majority of homozygous and a substantial proportion of heterozygous patients are refractory to conventional pharmacological therapy. Therapeutic options for these resistant patients are limited to LDL apheresis, portacaval anastomosis or liver transplantation.2 Gene therapy has been explored as an alternative treatment. Liver is the main target organ for FH gene therapy because of its capacity to dispose excess cholesterol by diverting it into bile acids; it is also accessible to gene delivery via the intravenous (i.v.) route or the hepatic artery. A number of studies have shown that hepatic reconstitution of LDLR expression ex vivo can reverse hypercholesterolemia, including promising results in a rabbit model of FH. 3 In the only clinical gene therapy trial for FH to date, Grossman et al.4, 5 isolated hepatocytes from FH patients, transduced them ex vivo with retroviral vector expressing LDLR and reimplanted them into the liver of the patients. Only marginal therapeutic benefit was achieved in this study. It was difficult to determine whether the reduction in LDL-C level was the direct result of the gene transfer or other factors were involved. Plasma LDL level is determined by LDL production and removal. For example, the decline of LDL-C after portacaval anastomosis is caused by a decreased secretion of very-low-density lipoprotein, a precursor of LDL, not by an enhanced LDL removal.6 In this clinical trial, LDL turnover was not measured, which led to the comment a modest 17% fall in plasma cholesterol after 25% hepatectomy and re-infusion of hepatocytes infected with a retrovirus might have been due to either diminished lipoprotein production or to enhanced activity of the patients own receptor.7 The focus has shifted to in vivo gene therapy thereafter. Helper-dependent adenoviral vector (HDAd) is devoid of all viral protein genes and has shown considerable promise for liver-directed gene transfer with long-term transgene expression, which lasted a lifetime in mice.8 In a previous study in LDLR/ mice, we showed that a single injection of HDAd expressing monkey LDLR reduced plasma cholesterol over 2 years and attenuated atherosclerotic lesion progression. 9 We also demonstrated that LDLR gene therapy induces the regression of established atherosclerosis in LDLR/ mice.10 Despite promising results of gene therapy in small animal models, its efficacy in large animal models has not been tested; there are important differences in physiology and in immune responses between rodents and humans. This issue is particularly relevant in gene therapy for lipid disorders.11

A nonhuman primate model of FH has been described in rhesus monkeys,12, 13 which carried a heterozygous nonsense mutation involving codon Trp283 14 of the LDLR. Extensive cross-breeding of the affected monkeys failed to yield any homozygotes, indicating that the mutation may be linked to a lethal mutation. With the availability only of the heterozygous (LDLR+/) rhesus monkey, we will be modeling heterozygous FH in humans, a relatively common genetic disorder that affects about 1 in 500 people in most ethnic groups.15 Heterozygous LDLR-deficient monkeys displayed elevated plasma cholesterol (5.176.47mmoll1 or 200250mgdl1) compared with unaffected monkeys (2.593.36mmoll1 or 100130mgdl1); the plasma cholesterol level further increased to 12.9320.69mmoll1 (500800mgdl1) when the animals were fed a high-cholesterol diet.16 In this study, we tested the efficacy of HDAd-based monkey LDLR gene therapy in high-cholesterol diet-fed LDLR+/ rhesus monkeys. We compared the effect of i.v. injection of HDAd-LDLR with that of a balloon catheter-based procedure developed by Brunetti-Pierri et al. 17 We found that a single i.v. injection of HDAd-LDLR into LDLR+/ monkeys produced a >50% lowering of plasma cholesterol that lasted about a month. We next tested a modified percutaneous catheter-based gene delivery strategy also developed by Brunetti-Pierri et al. 18 In this refinement, the HDAd-LDLR was injected directly into the hepatic artery in the presence of increased intrahepatic pressure induced by transient blockage of hepatic venous drainage by a balloon catheter positioned in the inferior vena cava (IVC). The optimized gene delivery strategy was highly efficacious in reducing the vector dose while substantially prolonging the therapeutic hypocholesterolemic response to the treatment regimen.

We treated four LDLR+/ monkeys as study subjects with a single i.v. injection of escalating doses of HDAd-LDLR. 9 We first treated monkey #8796 with 20ml of saline and found no significant changes in plasma cholesterol levels after treatment (Figure 1). As expected, we also failed to detect any change in plasma cholesterol when we treated another LDLR+/ monkey #9908 with an empty vector HDAd-0 (0.8 1012 viral particles (vp)kg). We next injected i.v. HDAd-LDLR into a third LDLR+/ monkey #7139 at a dose of 1.1 1012vpkg1, an HDAd dose that is 10-fold higher than the dose of HDAd--fetoprotein that stimulated significant elevation in -fetoprotein secretion in serum in baboons,17 and again failed to observe any change in plasma cholesterol level. We then treated a fourth monkey #13090 at an even higher i.v. dose of 5 1012vpkg1 of HDAd-LDLR. The treatment was well tolerated by the monkey and led to a 60% reduction in plasma cholesterol from a baseline of 14.95mmoll1 (578mgdl1) to 5.90mmoll1 (229mgdl1) on day 7. The plasma cholesterol lowering persisted until day 21, when it went up to 10.70mmoll1 (413mgdl1) on day 28, and toward pre-treatment levels on day 42. These results indicate that a dose higher than 1.1 1012vpkg1 was needed to reverse hypercholesterolemia in LDLR+/ monkeys, and a dose of 5 1012vpkg1 significantly restored normal plasma cholesterol in a heterozygous FH monkey, an effect that lasted for about a month. We next treated a fifth monkey #11226 with an even higher dose of 8.4 1012vpkg1, which was modestly below a dose that had previously proven to be lethal, 19 and observed severe acute toxicity and lethality within a day of treatment. The clinical picture and necropsy revealed hemorrhagic shock syndrome likely resulting from the high dose of HDAd vector used.

Efficacy of intravenous injection of HDAd expressing monkey LDLR in heterozygous LDLR-deficient rhesus monkeys. Four heterozygous LDLR-deficient monkeys were treated with a single intravenous injection of saline (#8796), empty vector at a dose of 0.8 1012vpkg1 (#9908) or HDAd-LDLR at a dose of 1.1 1012vpkg1 (#7139) or 5 1012vpkg1 (#13090). Baseline cholesterol levels were 18.0mmoll1 (696mgdl1) in monkey #8796, 9.5mmoll1 (368mgdl1) in monkey #9908, 8.0mmoll1 (308mgdl1) in monkey #7139 and 15.0mmoll1 (578mgdl1) in monkey #13090. The broken line shows pre-treatment cholesterol levels.

To improve on i.v. vector injection as a delivery method, Brunetti-Perri et al. developed a protocol 17, 18 to deliver the vector via an intrahepatic arterial catheter. Simultaneously, under fluoroscopic guidance, they inserted a balloon catheter into the IVC via the femoral vein and positioned it over the hepatic venous outflow (Figure 2a). Intrahepatic arterial HDAd injection when the balloon was inflated led to a 10-fold increase in efficiency in transgene expression ( Figures 2b and c). The IVC occlusion was also monitored by the venous pressure (Figure 2d). We performed the same procedure in rhesus monkeys and injected the HDAd vector (2ml) within a minute via a hepatic artery catheter immediately after the balloon was inflated.

Balloon catheter-based hepatic artery injection. (a) Schematic diagram of hepatic artery injection. Liver circulation is isolated by inserting a balloon catheter via the femoral vein and placing it in the IVC. A second intra-arterial catheter is inserted into the hepatic artery through the contralateral femoral artery. The placement of the catheter is visualized using fluoroscopy. Once occlusion of the hepatic circulation has been established via the balloon catheter in the IVC, the vector is injected via the arterial catheter. The occlusion is confirmed by monitoring hepatic venous pressure through the third catheter inserted into the femoral vein. BD, bile duct; HA, hepatic artery; HV, hepatic vein; PV, portal vein. (b) Fluoroscopy image to confirm the position of a balloon catheter. (c) Fluoroscopy after the balloon inflated. Contrast reagent was injected to confirm that the catheter was placed at the IVC. (d) Venous pressure. Occlusion was monitored by venous pressure.

The monkeys used for this procedure are summarized in Table 1. We first performed the procedure in a chow-fed (Purina LabDiet5LEO, St Louis, MO, USA) normal LDLR+/+ (#19254) and a heterozygous LDLR+/ (#19499) monkey. The injection was done immediately after the balloon was deflated but while hepatic venous pressure remained high. As reported previously, 17,18 systemic blood pressure fell significantly when the balloon was inflated. We found that serum interleukin (IL)-6 level increased 30min after injection and peaked at 2h ( Figure 3a) but decreased to non-detectable levels by 72h. The procedure also led to transient and inconsistent changes in plasma liver enzymes ( Figures 3b and c). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels peaked at about 24h; the increase was mild and resolved by day 5. Plasma total cholesterol levels in the LDLR+/ (#19499) monkey decreased from a baseline of 5.70mmoll1 (219mgdl1) to 3.90mmoll1 (150mgdl1) within 24h. It gradually went back up over the next few days returning to baseline by day 5. The plasma cholesterol level did not change in the non-FH (LDLR+/+) (#19254) monkey ( Figure 3d).

Acute toxicity measurements associated with balloon catheter-based hepatic artery injection. One normal LDLR+/+ (#19254) and one heterozygous LDLR+/ (#19499) monkeys on normal chow were treated by an injection of saline and a complete blood test and IL-6 measurement were performed. (a) Plasma IL-6 levels. (b) Serum ALT levels. (c) Serum aspartate aminotransferase (AST) levels. (d) Plasma cholesterol levels.

We next fed monkeys with a rhesus Western diet (Texas Biomedical Research Institute, San Antonio, TX, USA) for 7 weeks before treatment and were kept on the diet afterward. We injected HDAd-LDLR (2 1012vpkg1) into four monkeys immediately after the balloon was deflated. The plasma cholesterol did not change in two wild-type LDLR+/+ monkeys (#19360 and #21588) suggesting that the gene delivery does not have an effect on the cholesterol dynamics in monkeys that express normal amounts of LDLR. Of the two heterozygous LDLR+/ monkeys, one (#19251) showed no change in plasma cholesterol ( Figure 4a, green line), whereas another LDLR+/ monkey (#19498) exhibited a 57% drop in plasma cholesterol level from 8.15mmoll1 (315mgdl1) to 3.25mmoll1 (126mgdl1) at day 7 ( Figure 4a, red line). So there was a heterogeneous response in heterozygous FH monkeys treated at this dose of HDAd-LDLR. The cholesterol-lowering effect of HDAd-LDLR in the LDLR+/ (#19498) monkey that responded to the treatment was sustained for about 100 days. The plasma-lowering effect reached its nadir 7 days, and stayed at or near the nadir for another 3 weeks. Afterward, it gradually rose to 5.09mmoll1 (197mgdl1) at day 78, and then to above the pre-treatment level (9.30mmoll1 or 361mgdl1) by day 105 ( Figure 4a, red line). The two wild-type LDLR+/+ monkeys maintained normal serum ALT throughout the observation period of 120 days. The LDLR+/ monkey (#19251) that did not show a hypocholesterolemic response also maintained normal ALT levels for 67 days, end of the observation period for this monkey. In contrast, the serum ALT of the LDLR+/ monkey (#19498) that showed a hypocholesterolemic response maintained a normal ALT level during the first 3 weeks of treatment when the plasma cholesterol showed an excellent response ( Figure 4a, red line). ALT began to edge above normal to 70Ul1 on day 36, and continued to go up to peak at 144Ul1 on day 72, before it started trending down, eventually returning to normal on day 120 ( Figure 4b, red line). It is noteworthy that this monkey that had responded to the treatment developed liver enzyme elevation late, and the delayed increase in serum ALT coincided with the onset of loss of the cholesterol-lowering effect of the treatment. Although the significance of the timing is unclear, we note that a similar pattern is evident in an experiment involving another LDLR+/ monkey (#19269, see below).

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Gene Therapy - Gene therapy for rhesus monkeys ...

Drugmakers have a slew of treatments for afflictions related to sex and drugs. Now they may have one for rock n roll.

Novartis AG (NOVN) is developing a gene therapy that may reverse hearing loss by stimulating the regrowth of microscopic hair cells in the inner ear, allowing people to hear. The hairs are destroyed by prolonged exposure to loud noise, and dont take root again naturally. Novartis treated the first patient in October after successful tests on rats.

While hearing loss is most common in the elderly, rates are high in the music industry and the military, and rising among teenagers who listen to music at high volume. Almost 13 percent of children and adolescents under 19 in the U.S. have permanent damage caused by excessive exposure to noise, according to the Centers for Disease Control and Prevention.

A little too much Lady Gaga, said Mark Fishman, the head of Novartis Institutes for BioMedical Research, which is developing the therapy. About 36 million people in the U.S. have some form of hearing loss, according to the Basel, Switzerland-based company.

A solution could mean big money for Novartis and GenVec Inc. (GNVC), its partner in developing the drug. Global sales of hearing aids and cochlear implants may reach a combined $9.5 billion globally by 2020, according to San Francisco-based Grand View Research, which provides information on industries including technology and health care.

Global sales of hearing aids and cochlear implants may reach a combined $9.5 billion globally by 2020, according to San Francisco-based Grand View Research, which provides information on industries including technology and health care. Close

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Global sales of hearing aids and cochlear implants may reach a combined $9.5 billion globally by 2020, according to San Francisco-based Grand View Research, which provides information on industries including technology and health care.

Novartis plans to test its treatment on 45 patients in the U.S., with results expected by 2017, according to a description of the trial on clinicaltrials.gov, the National Institutes of Healths database of studies. Its too early to say when the treatment might be approved, Fishman said.

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Lady Gaga at High Volume Drives Hearing-Loss Drug Search: Health

Genome Project legacy: advancements in gene th
KFDM #39;s Six On Health, sponsored by Christus Hospital St. Elizabeth #39;s and St. Mary explores how they Human Genome Project #39;s effort to map our chromosomal makeup has led to advances in gene ...

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Genome Project legacy: advancements in gene th - Video

HOPE, HYPE REALITY: TERMS USED IN GENE THERAPY

By: defeatHIV

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HOPE, HYPE & REALITY: TERMS USED IN GENE THERAPY - Video

Published January 07, 2015

Novartis is diving deeper into the world of gene-based medicine by signing deals with two U.S. biotech companies, giving it access to a powerful new genome editing technology.

The tie-ups with unlisted Intellia Therapeutics and Caribou Biosciences show the Swiss drugmaker's confidence in the potential of so-called CRISPR technology, both for making new medicines and as a research tool.

CRISPR, which stands for clustered regularly interspaced short palindromic repeats, allows scientists to edit the genes of selected cells accurately and efficiently. It has created great excitement since emerging two years ago and is being tipped for a Nobel Prize.

While current gene therapy approaches involve adding genes to affected cells, CRISPR opens up the possibility of correcting those cells' faulty genes in the lab before returning them to the patient.

Translating that promise into new treatments will take many years but Novartis' decision to apply the technology in its research labs is an important endorsement, since the company is the world's largest drugmaker by sales.

It is also a sign the Swiss group intends to be at the forefront of the nascent field, after recently establishing a new cell and gene therapies unit within the company.

Mark Fishman, head of the Novartis Institutes for BioMedical Research (NIBR), said genome editing could open a new branch of medicine, leading to cures for diseases caused by faulty genes.

"We have glimpsed the power of CRISPR tools in our scientific programmes in NIBR and it is now time to explore how to safely extend this powerful technology to the clinic," he said.

The deal with Intellia gives Novartis exclusive rights to develop programmes focused on engineered chimeric antigen receptor T-cells (CARTs) and the right to develop a certain number of targets for editing hematopoietic stem cells.

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Novartis taps into gene editing for next generation drugs

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History explains why people with the malady, and their physicians, are cautious to believe that a cure is in sight

HEATHER VAN UXEM LEWIS

In 2011, a remarkable study in the New England Journal of Medicine detailed the successful treatment of six adults with haemophilia B, which is caused by a deficiency in the coagulation protein known as factor IX. All of the participants were able to eliminate or reduce the frequency of clotting-factor-replacement injections the current standard treatment for the disease after their livers began producing functional levels of factor IX. The experimental therapy came in the form of an adeno-associated virus (AAV) carrying a gene that encodes instructions for production of normal levels of human factor IX. Three trials of AAV-mediated gene transfer in patients with haemophilia B are ongoing, with high expectations.

After more than 20 years of research on gene transfer, it is a promising time for haemophilia therapies. It now seems likely that a single-dose treatment for haemophilia B using an AAV or another gene-transfer technique will be a viable option for many people in the next decade or two.

Yet haemophilia researchers are not inclined to speak enthusiastically of a cure. Part of that caution comes from recognition that there are still problems to solve. For example, some 40% of people with haemophilia B would find no refuge in an AAV treatment because they produce antibodies that attack and neutralize this virus.

And even if that problem were solved, the treatment would apply only to those with haemophilia B. The more common form of the condition, haemophilia A, stems from a deficit in another protein factor VIII and the gene for that protein is a more difficult target. Regardless of the type of haemophilia, researchers remain hesitant about gene therapy owing to the unresolved ethical issues that arose decades ago.

The unfettered optimism that characterized the early years of gene-therapy research came to a screeching halt in 1999, when 18-year-old Jesse Gelsinger died in a phase I clinical trial at the University of Pennsylvania in Philadelphia. Gelsinger had undergone an experimental gene transfer for his otherwise treatable metabolic disorder. His death, along with a series of other harmful events in early gene-therapy trials for a variety of diseases, threatened the whole field.

Haemophilia specialists who were engaged in gene-transfer studies were more guarded than most of that era's self-proclaimed gene doctors. The source of their reserve goes beyond the cautious optimism that characterized such research after 1999; it is grounded instead in the long and troubled experience that the haemophilia community has had with technological fixes.

By the late 1970s, a therapeutic revolution had transformed haemophilia from an obscure hereditary malady into a manageable disease. But the glory of this achievement was tragically short-lived. The same clotting-factor-replacement therapies that delivered a degree of normality to the lives of people with haemophilia brought unexpected and fatal results: tens of thousands of people with haemophilia were diagnosed with transfusion-related HIV/AIDS in the 1980s and with hepatitis C virus (HCV) in the 1990s.

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Gene Therapys Hemophilia Promise Is Tempered by Memories of Past Tragedies

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History explains why people with the malady, and their physicians, are cautious to believe that a cure is in sight

HEATHER VAN UXEM LEWIS

In 2011, a remarkable study in the New England Journal of Medicine detailed the successful treatment of six adults with haemophilia B, which is caused by a deficiency in the coagulation protein known as factor IX. All of the participants were able to eliminate or reduce the frequency of clotting-factor-replacement injections the current standard treatment for the disease after their livers began producing functional levels of factor IX. The experimental therapy came in the form of an adeno-associated virus (AAV) carrying a gene that encodes instructions for production of normal levels of human factor IX. Three trials of AAV-mediated gene transfer in patients with haemophilia B are ongoing, with high expectations.

After more than 20 years of research on gene transfer, it is a promising time for haemophilia therapies. It now seems likely that a single-dose treatment for haemophilia B using an AAV or another gene-transfer technique will be a viable option for many people in the next decade or two.

Yet haemophilia researchers are not inclined to speak enthusiastically of a cure. Part of that caution comes from recognition that there are still problems to solve. For example, some 40% of people with haemophilia B would find no refuge in an AAV treatment because they produce antibodies that attack and neutralize this virus.

And even if that problem were solved, the treatment would apply only to those with haemophilia B. The more common form of the condition, haemophilia A, stems from a deficit in another protein factor VIII and the gene for that protein is a more difficult target. Regardless of the type of haemophilia, researchers remain hesitant about gene therapy owing to the unresolved ethical issues that arose decades ago.

The unfettered optimism that characterized the early years of gene-therapy research came to a screeching halt in 1999, when 18-year-old Jesse Gelsinger died in a phase I clinical trial at the University of Pennsylvania in Philadelphia. Gelsinger had undergone an experimental gene transfer for his otherwise treatable metabolic disorder. His death, along with a series of other harmful events in early gene-therapy trials for a variety of diseases, threatened the whole field.

Haemophilia specialists who were engaged in gene-transfer studies were more guarded than most of that era's self-proclaimed gene doctors. The source of their reserve goes beyond the cautious optimism that characterized such research after 1999; it is grounded instead in the long and troubled experience that the haemophilia community has had with technological fixes.

By the late 1970s, a therapeutic revolution had transformed haemophilia from an obscure hereditary malady into a manageable disease. But the glory of this achievement was tragically short-lived. The same clotting-factor-replacement therapies that delivered a degree of normality to the lives of people with haemophilia brought unexpected and fatal results: tens of thousands of people with haemophilia were diagnosed with transfusion-related HIV/AIDS in the 1980s and with hepatitis C virus (HCV) in the 1990s.

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Gene Therapys Haemophilia Promise Is Tempered by Memories of Past Tragedies

CHOP gene therapy expert fine-tunes protein signals, improves motor function and reduces brain shrinkage in a neurological disorder

IMAGE:Beverly L. Davidson, Ph.D., a gene therapy expert, is the director of The Center for Cellular and Molecular Therapeutics at the Children's Hospital of Philadelphia. view more

Credit: The Children's Hospital of Philadelphia

By adjusting the levels of a key signaling protein, researchers improved motor function and brain abnormalities in experimental animals with a form of Huntington's disease, a severe neurodegenerative disorder. The new findings may lay the groundwork of a novel treatment for people with this fatal, progressive disease.

"This research shows the intricate workings of a biological pathway crucial to the development of Huntington's disease, and is highly relevant to drug development," said study leader Beverly L. Davidson, Ph.D., director of The Center for Cellular and Molecular Therapeutics at The Children's Hospital of Philadelphia (CHOP). "Our results in animals open the door to a promising potential therapy, based on carefully manipulating the dysregulated pathway to treat this devastating human disease."

She added that restoring proper balance to these delicate biological processes may offer even broader benefits in treating other neurological diseases, such as amyotrophic lateral sclerosis (ALS), fragile X mental retardation and autism.

The study team published its results online Dec. 31 in the journal Neuron.

Huntington's disease is an incurable, inherited disease entailing progressive loss of brain cells and motor function, usually beginning in midlife. A defective gene produces repeated copies of a protein called huntingtin, or HTT. The mutant HTT protein (mHTT) particularly damages a brain region called the striatum, where it interferes with normal cell growth and other fundamental biological events. The resulting disease includes involuntary movements and severe cognitive and emotional disturbances. About 30,000 Americans have Huntington's disease (HD).

Neuroscientists already knew that a signaling protein called mTORC1 that regulates cell growth and metabolism plays a major role in HD. Many researchers have proposed that inhibiting or shutting off the mTORC1 pathway, which interacts with the deleterious mHTT proteins, could help treat HD.

The current study contradicts those assumptions. "We show that the mTORC1 pathway is already impaired in Huntington's disease, and that improving how the pathway functions actually has a protective effect," said Davidson. "However, restoring that pathway must be done very carefully to avoid further harm. It's a 'Goldilocks effect.' You need to restore the mTORC1 level; either too much or too little is detrimental."

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Animal study points to a treatment for Huntington's disease

Preliminary Results from Gene Therapy in Patients with Beta Thalassemia
At the American Society of Hematology Annual Meeting, preliminary data using bluebird bio #39;s gene therapy to treat beta thalassemia and sickle cell anemia was...

By: Rare Disease Report

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Preliminary Results from Gene Therapy in Patients with Beta Thalassemia - Video

Rhys Evans life could have been very different.

He could have been a bubble boy, forced to walk around in a protective see-through plastic canopy. You see, he was born with an immune system that barely worked. The slightest infection could have proved fatal. But Rhys is now 14years old and doing fine.

So how did Rhys avoid living in a bubble?

The simple answer is that Rhys got lucky his condition was diagnosed when he was a baby. Even more fortunately, doctors at Great Ormond Street Hospital were able to do something about it. They understood that Rhyss condition was caused by a genetic flaw and they thought that if they could correct this flaw then they could restore his immune system. That is exactly what happened, and why Rhys is now no different to any other young teenager.

Rhyss treatment is an example of gene therapy, which was the subject of a fascinating lecture that I attended last month. Leonard Seymour, professor of gene therapies in the Department of Oncology at Oxford University, gave four reasons why 2014 has been a breakthrough year for this revolutionary, but controversial, approach.

Let me begin by describing these successful trials.

Rhys Evans is not the only boy (it does not affect girls) to have received gene therapy for this syndrome 20 were given it at about the same time as Rhys. But he was lucky. In the trial, one in four ended up with leukaemia.

This year has seen the results of a new trial. In this, nine boys were treated and eight have been reported as still alive, 16 to 43 months after treatment. The ninth died from an infection already present when he began the gene therapy. Overall, the outcome is hugely promising and suggests that gene therapy could provide a permanent cure for patients who would otherwise receive a bone marrow transplant from a donor, with all the consequent risks of rejection.

HIV is a virus that weakens the immune system by destroying the white blood cells that fight disease and infection. In order to destroy the cells it has to enter them, and it does this via a protein called CCR5, found on the cell surface. Researchers have noticed that about 1% of patients contract HIV and yet come to no harm. The reason is that their cells have a rare genetic mutation which prevents them from displaying the CCR5 protein on their surface.

Now researchers have managed to engineer white blood cells so that they have this same rare mutation. They have injected billions of these genetically modified cells into 12 trial patients, and there is evidence that this procedure is safe and could suppress the virus.

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Why 2014 has been a breakthrough year for gene therapy

The year in biotechnology began with a landmark event. A decade after the first human genome was decoded at a cost of about $3 billion, the sequencing-machine company Illumina, of San Diego, introduced a new model, the Hyseq X-10, that can do it for around $1,000 per genome.

The system, which costs $10 million and can decode 20,000 genomes a year, was snapped up by large research labs, startup firms like J. Craig Venters Human Longevity (which plans to sequence 40,000 people a year), and even by the British government (the U.K. is the first country with a national genome sequencing project).

Francis de Souza, Illuminas president, predicted that within two years the genomes of about 1.6 million people will have been sequenced.

Cheap sequencing means a deluge of information and a new role for technology designed to handle and exploit big data. The search giant Google was the tech company most attuned to the trend, launching a scientific project to collect biological data about healthy humans, and offering to store any genome on its servers for $25 per year. A coalition of genetics researchers backed by Google tried to introduce technical standards, like those that govern the Web, as a way of organizing an Internet of DNA over which researchers might share data.

Easy access to DNA information led to debates over how much consumers should know. The U.S. Food and Drug Administration has said direct-to-consumer genetic health tests arent yet ready to be marketed. But consumers found ways to get the data anyway. Thousands of people headed to unregulated corners of the Internet to learn about their genes, and one father even managed to sequence the DNA of his own unborn son, claiming a controversial first.

Easily the hottest technology of the year was a new gene-engineering method called CRISPR, a powerful new editing system for DNA. Chinese scientists used it to produce genetically altered monkeys in January, and other scientists are now expected to create monkeys that model human psychiatric diseases. One measure of the technologys importance is that scientists are now fighting over who really invented it firstand who should own the patent on it.

During the year, bioengineers advanced on all fronts using other technologies. We saw novel kinds of cell therapy used to treat degenerative eye diseases, positive results from a study of gene therapy that could cure HIV, and the resurgence of a form of gene therapy called RNA interference. The development of replacement organs took steps forward, too, including new research showing how to add blood vessels to lab-made tissue using a 3-D printer.

This year, 10 of 35 new drugs approved by the FDA were biological molecules, like antibodies or protein injections. That was a record. And the FDA says the list of new drugs entering testing for the first time is dominated by biological treatments.

Those biotech drugs include the most important medical breakthroughs of the year, a new class of cancer drugs called immunotherapies. The drug company Merck has been testing an antibody that helps the immune system recognize melanoma cancer cellswith near miraculous results for some patients. The other approach to immune therapy involves rengineering a persons white blood cellsto recognize and kill certain kinds of leukemia tumors.

Bioengineering doesnt stop at DNA. The U.S. BRAIN Initiative, President Obamas signature science project, has the aim of developing emerging neurotechnologies for measuring the brain and eventually figuring out the neural code. The broad approach of the U.S. project contrasts with that taken in Europe, where funding has been directed toward one mega-project to create computer simulations of the brain, something that drew sharp fire from dissenting neuroscientists.

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2014 in Biomedicine: Rewriting DNA, Decoding the Brain, and a GMO Paradox