Category Archives: Genetic Engineering

This two-part review provides high-level analytical development considerations for exvivo, genome-modified hematopoietic stem and progenitor cell (GM-HSPC) products derived from primary donor cells. Part 1 in BPIs May 2024 issue addresses analytical controls for in-process drug substances and drug products. Here in Part 2, we take a step back to examine concerns for HSPC source materials. Look to other recently published reviews for a broader discussion of chemistry, manufacturing, and controls (CMC) for GM-HSPCs (19, 20) and for development considerations with gene-edited pluripotent stem cells (PSCs) (21). Note that we use the term genome modified in a generic sense herein to include products that are manufactured by means of viral-vector transduction (typically by lentiviral vectors (LVVs)) and those subject to genome editing by such means as a system based on clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9).

Analytical Controls for Starting Materials and Drug Substances

To ensure GM-HSPC quality, thorough analytical control strategies should be implemented that include a phase-appropriate set of in-process, characterization, and release tests to monitor both manufacturing processes and drug products. Figure 2 depicts a generic GM-HSPC manufacturing process, beginning with the introduction of cellular starting material, which is controlled through establishment of donor eligibility criteria and starting-material testing. Genome-modification reagents e.g., nucleases, single-guide RNA (sgRNA), and viral vectors usually are classified as drug substances and thus are subject to release testing before their entry into the manufacturing process. Control of both cellular starting material and genome-modification reagents are discussed below. Note that GM-HSPC manufacturing processes often proceed uninterrupted, and often there is minimal or no testing of cellular drug substances.

Figure 2: Standard manufacturing process for genome-modified hematopoietic stem and progenitor cell (GM-HSPC) therapies. sgRNA = single-guide ribonucleic acid.

Many starting materials and critical reagents for products such as GM-HSPCs can be of varying quality and/or the product of bespoke manufacturing, themselves. Thus, it is necessary to place significant emphasis on the analysis of cellular starting materials and genome-modification reagents. For these complex products, investing up front in a comprehensive analytical approach might help to accelerate development and mitigate later-stage risks.

Cellular Starting Materials: Most GM-HSPC processes including those used to make the six commercially approved products to date (see Part 1, Table 1) rely on the acquisition of autologous cellular starting material (6671). Allogeneic products have entered clinical development recently (72). Such efforts include ongoing work to establish universal HSPCs derived from PSCs. Those products are beyond the scope of this review; considerations for their development were described by Morse and Mack in 2023 (21).

Whether derived from an autologous or allogeneic source, control of cellular starting material follows two parallel pathways: donor eligibility determination and cellular testing (Table 3). Questionnaires, medical examinations, and viral testing serve to establish donor eligibility (73, 74). Donor testing typically includes testing for hepatitis, human immunodeficiency virus (HIV), and other pathogens as well as prion contagions. After those screenings, eligible donors are subjected to a mobilization regimen that enables CD34+ stem cells to move from the donors bone marrow to their peripheral blood (75, 76). Thus mobilized, they provide peripheral-blood donation that is subjected to leukapheresis, extraction of white blood cells from the peripheral blood (9). Alternatively, CD34+ stem cells may be acquired by extraction of bone marrow directly from the hip bone (9).

Table 3: Control of cellular starting materials includes both assessment of donor eligibility and testing of those materials. Minimal donor eligibility considerations are provided below; for a more extensive discussion, see reference 74. Abbreviations: CJD = CreutzfeldtJakob disease; HIV = human immunodeficiency virus; HTLV = human T-lymphotropic virus; HBV = hepatitis B virus; HCV = hepatitis C virus; TSE = transmissible spongiform encephalopathy; WNV = West Nile virus.

Cellular testing begins upon receipt of leukopaks or other starting material at the biomanufacturing site. Such testing typically focuses on the quantity and health of target CD34+ cells through testing of viability and measurement of both total nucleated cell count and the percentage of CD34+ cells present. When possible, limits for such tests should be based on manufacturing capability and potential effects on critical quality attributes (CQAs). Limits are likely to be established based on a minimum viability to provide a product of sufficient quality and a minimal cell count and CD34+ percentage to ensure adequate dosing. Additional tests e.g., safety testing or analysis of specific cell phenotypes also might be considered necessary for a GM-HSPC program.

Precedents have been established for several approaches to genetic modification of HSPCs (77). For our purposes, we consider genome-modification reagents including viral vectors, nucleases, and sgRNA to be active pharmaceutical ingredients (APIs) or drug substances and therefore subject to an appropriate level of analytical control (78). Release of each reagent should be contingent upon demonstration of sufficient purity, safety, and potency.

Nucleases: Release specifications for nucleases such as the Cas9 protein should be set to ensure both the consistency of the manufactured nuclease and the safety, purity, and potency of the corresponding GM-HSPC drug product. Table 4 lists typical assays for nuclease release. The list is not exhaustive and applies only to the given nuclease (protein) itself, although similar principles apply to mRNA as well.

Table 4: Typical assays included in a nuclease-release assay panel. Abbreviations: ELISA = enzyme-linked immunosorbent assay; HPLC = high-performance liquid chromatography; qPCR = quantitative polymerase chain reaction; SEC = size-exclusion chromatography; USP = United States Pharmacopeia.

Perhaps the most important nuclease attributes are safety and activity, which are key to ensuring quality of GM-HSPC drug products. Safety test panels include compendial sterility, mycoplasma, and endotoxin assays (or equivalents); activity assays might be designed to measure the ability of a nuclease to cut (or otherwise modify) template DNA. Evaluation of purity typically requires a method such as high-performance liquid chromatography (HPLC) to measure the percentage of intact, full-length nuclease molecules. Impurity determination often relies on multiple assays to evaluate host-cell proteins (HCPs), host-cell DNA, and nuclease degradants/aggregates present in a product sample.

An increasing number of good manufacturing practice (GMP)quality nucleases have become available commercially, providing a useful route to minimizing cost and complexity relative to internal manufacturing (7981). However, to ensure that suppliers can support GM-HSPC programs throughout development, product sponsors should exercise appropriate oversight (e.g., vendor management programs, audits, and so on) before integrating off-the-shelf options. For early stage clinical development, that includes robust platform assays and specifications that are appropriate for manufacturing HSPC drug products. For later-stage development including studies enabling licensure of clinical material and commercial manufacturing sponsors should ensure that their suppliers have strong analytical validation programs in place. In such later phases, sponsors also need their own appropriate quality systems and risk-assessment procedures with associated documentation of all changes in production processes and analytical methods.

Single-Guide RNA: Given the critical nature of sgRNA sequences in determining CRISPR/Cas9 specificity, their purity and identity are considered to be critically important to PQAs. Ion-pair reversed-phase HPLC (IP-RP HPLC), which separates oligonucleotides based on their length and charge, is a standard method used for measuring sgRNA purity (81). Note, however, that both molecular length and commonly used chemical modifications such as phosphorothioate linkages can present significant challenges in the use of chromatographic approaches (82). Mass-spectrometry (MS) and next-generation sequencing (NGS) approaches also can be used for establishing sequence purity.

Recent publications demonstrate that LC-MS approaches can be used to demonstrate sequence identity, to detect sequence modifications, and possibly to establish the sequence purity of targeted regions of sgRNA molecules (8385). However, LC-MS has yet to sequence full-length sgRNA quantitatively. NGS analysis theoretically should apply to quantitative sequencing of sgRNA but for evaluation of chemical modifications, although biases during amplification can complicate the techniques reliability in quantitating sequences (86). Such effects should be evaluated before implementation of NGS assays for sgRNA purity assessment. Table 5 lists assays usually found on sgRNA release-testing panels.

Table 5: Typical assays included in a release-assay panel for sgRNA. Abbreviations: GC = gas chromatography; IP-RP HPLC = ion-pair reversed-phase high-performance liquid chromatography; LC-MS = liquid chromatographymass spectrometry; NGS = next-generation sequencing; ICP-MS = inductively coupled plasma mass spectrometry; sgRNA = singleguide ribonucleic acid; USP = United States Pharmacopeia.

Ribonucleoprotein (RNP): Neither sgRNA nor RNA-directed nucleases have significant biological activity in isolation. Rather, those components combine to form RNP complexes that modify DNA. Thus, regulators expect sponsors to provide at least some characterization of those complexes. For cases in which RNPs are prepared ex vivo, sponsors at minimum should implement both purity and activity assays to characterize those complexes. Purity assessments may include analysis of both the proportion of intact RNP and proportions of sgRNA and nuclease present (87); activity assays probably can be established in the same manner as noted above for the release of sgRNA or nuclease.

Viral Vectors: Lentiviral vector (LVV) manufacturing poses several challenges, including needs for consistent production (e.g., by stable and high-yielding producer cell lines) and for highly accurate titer assays for quantifying LVV concentrations and safety (8889). Unlike other viral vectors, LVV production poses difficulty to those companies attempting to use stable cell lines (90). Thus, most conventional practices use transient transfection of adherent cell lines, which has presented difficulties in scale-up (91). That said, establishing stable cell lines would eliminate steps required for transient transfection and enable continuous and consistent vector production (92).

Analytical control of LVVs involves a comprehensive and well-established set of methods and techniques for ensuring the quality, purity, and functionality of vector preparations. Several methods are used in quantifying viral titers to ensure the appropriate dosage for intended applications. Those methods can be categorized broadly into functional and nonfunctional approaches (93). Nonfunctional (physical) titer methods include assessments of p24 capsid protein and lentiviral RNA levels. A significant disadvantage of such methods is the potential for overestimating vector titers through quantification of protein or RNA coming from both functional and defective vector particles (93). Infectious titer assays use real-time quantitative polymerase chain reaction (RT-qPCR) to measure mRNA expression from transduced cells. Such a functional approach is considered to be more accurate for determination of functional titers (94).

Additional key aspects of analytical control include evaluating the absence of contaminants such as HCPs, nucleic acids, and other foreign particles; verification that LVVs have the correct genetic material and maintain their intended identity; assessment of the vectors ability to achieve desired transduction efficiency; and analysis of LVV integration patterns within a host genome to evaluate the risk of insertional mutagenesis (95). Table 6 is a typical release-testing panel for LVVs used in manufacturing GM-HSPCs.

Table 6: Standard viral vector release-assay panel. * In this context, dose-defining refers to the dose used in the manufacturing process. ** Residual impurities can include residual plasmid DNA (pDNA), host-cell proteins, host-cell DNA (potentially including E1A and SV40 DNA sequences), and encapsulated residual DNA (potentially including plasmids, host-cell DNA, E1A). Abbreviations: CE-SDS = capillary electrophoresissodium dodecyl sulfate; DLS = dynamic light scattering; ELISA = enzyme-linked immunosorbent assay; GM-HSPC = genetically modified hematopoetic stem and progenitor cells; LVV = lentiviral vector; qPCR = quantitative polymerase chain reaction; SDS-PAGE = sodium dodecyl sulfatepolyacrylamide gel electrophoresis; USP = United States Pharmacopeia.

With measures such as splitting of the LVV genome into separate plasmids and partial deletion of the 3' long-terminal repeat (LTR) reducing risks of replication competence, the safety of LVVs has improved over the years, thus minimizing the associated patient safety risks (96). The recommended assay for assessing replication-competency of viruses involves coculture with indicator cells and subsequent evaluation of the presence of viral protein and/or DNA sequences. Nevertheless, alternative rapid methods can be used for detecting replication-competent LVV if their equivalence or superiority to the traditional coculture assay can be demonstrated (97).

Rising to the Challenges

Whether manufactured through viral-vectormediated gene delivery, nuclease-mediated editing, or both, genome modification of HSPCs represents a significant advancement in the potential to cure diseases that otherwise have suboptimal or no currently available treatment options. Building on a substantial therapeutic legacy of HSC transplants, these new therapies are complex to manufacture and require broad and deep analytical support to ensure adequate and consistent product quality (13).

GM-HSPCs face two key analytical challenges: First, the components used to manufacture them are often bespoke and require significant analytical oversight. Second, as detailed in Part 1 of this review, the broad range of materials used to manufacture a GM-HSPC can include proteins, nucleic acids, viral vectors, and cellular materials. Each of those requires the development of its own bespoke analytical approach, including specific analytical tools and methods. Challenges associated with appropriate characterization of GM-HSPC products will increase as the field matures, with the potential addition of new gene-editing techniques such as base and Prime editing and gene writing (6264) as well as in vivo targeting approaches (65).

Thus, GM-HSPC sponsors are advised to put significant thought into the development of appropriate analytical control strategies for each of their candidate therapies. Doing so can help maximize the probability of regulatory, technical, clinical, and commercial success, thus helping to maximize the likelihood of each candidate achieving its therapeutic potential.

References

Note: See part 1 for References 165 (Morse B, Fiedorowicz A. Analytical Considerations for Gene-Modified Hematopoietic Stem and Progenitor Cell Therapies, Part 1 In-Process and Drug Product Controls. BioProcess Int. 22(5) 2024: 1723; https://www.bioprocessintl.com/cell-therapies/analytical-considerations-for-gene-modified-hematopoietic-stem-and-progenitor-cell-therapies-part-1-in-process-drug-substances-and-drug-products.

66 Strimvelis: EPAR Product Information. European Medicines Agency: Amsterdam, the Netherlands, 2016; https://www.ema.europa.eu/en/documents/product-information/strimvelis-epar-product-information_en.pdf.

67 Zynteglo Package Insert. US Food and Drug Administration: Silver Spring, MD, 2022; https://www.fda.gov/media/160991/download.

68 Libmeldy: EPAR Product Information. European Medicines Agency: Amsterdam, the Netherlands, 2020; https://www.ema.europa.eu/en/documents/product-information/libmeldy-epar-product-information_en.pdf.

69 Skysona Package Insert. US Food and Drug Administration: Silver Spring, MD, 2022; https://www.fda.gov/media/161640/download.

70 Lyfgenia Package Insert. US Food and Drug Administration: Silver Spring, MD, 2023; https://www.fda.gov/media/174610/download.

71 Casgevy Package Insert. US Food and Drug Administration: Silver Spring, MD, 2023; https://www.fda.gov/media/174615/download.

72 Lydeard JR, et al. Development of a Gene-Edited Next-Generation Hematopoietic Cell Transplant To Enable Acute Myeloid Leukemia Treatment by Solving Off-Tumor Toxicity. Mol. Ther. Meth. Clin. Dev. 13(31) 2023: 101135; http://doi.org/10.1016/j.omtm.2023.101135.

73 CBER. Guidance for Industry: Implementation of Acceptable Full-Length and Abbreviated Donor History Questionnaires and Accompanying Materials for Use in Screening Donors of Blood and Blood Components. US Food and Drug Administration: Silver Spring, MD, 2023, https://www.fda.gov/media/124193/download.

74 CBER. Guidance for Industry: Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps). US Food and Drug Administration: Silver Spring, MD, 2007; https://www.fda.gov/media/73072/download.

75 Fresen MM, et al. Stem Cell Mobilization With and Without Plerixafor: A Comparative Analysis. J. Hematol. Blood Transf. Disord. 5(1) 2018: 100018; https://doi.org/10.24966/HBTD-2999/100018.

76 Tisdale JF, et al. Single-Agent Plerixafor Mobilization To Collect Autologous Stem Cells for Use in Gene Therapy for Severe Sickle Cell Disease. Transpl. Cell. Ther. 24(3) 2018: S174; http://doi.org/10.1016/j.bbmt.2017.12.108.

77 Ferrari S, et al. Genetic Engineering Meets Hematopoietic Stem Cell Biology for Next-Generation Gene Therapy. Cell Stem Cell 30(5) 2023: 549-570; http://doi.org/10.1016/j.stem.2023.04.014.

78 CBER. Guidance for Industry: Human Gene Therapy Products Incorporating Human Genome Editing. US Food and Drug Administration: Silver Spring, MD, 2024; https://www.fda.gov/media/156894/download.

79 Gene Editing Enzymes. Aldevron: Fargo, ND, 2023; https://www.aldevron.com/catalog-products/nucleases.

80 cGMP Cas9 Nuclease. Akron Biotech: Boca Raton, FL, 2024; https://akronbiotech.com/product/nls-spcas9-nls-nuclease-solution.

81 GMP Recombinant Cas9. Takara Bio USA: San Jose, CA, 2024, https://www.takarabio.com/products/gene-function/gene-editing/crispr-cas9/gmp-recombinant-cas9.

82 Donegan M, Nguyen JM, Gilar M. Effect of Ion-Pairing Reagent Hydrophobicity on Liquid Chromatography and Mass Spectrometry Analysis of Oligonucleotides. J. Chromatogr. A 1666, 2022: 462860; https://doi.org/10.1016/j.chroma.2022.462860.

83 Gilar M, Koshel BM, Birdsall RE. Ion-Pair Reversed-Phase and Hydrophilic Interaction Chromatography Methods for Analysis of Phosphorothioate Oligonucleotides. J. Chromatogr. A 1712, 2023: 464475; https://doi.org/10.1016/j.chroma.2023.464475.

84 Goyon A, et al. Full Sequencing of CRISPR/Cas9 Single Guide RNA (sgRNA) via Parallel Ribonuclease Digestions and Hydrophilic Interaction Liquid Chromatography-High-Resolution Mass Spectrometry Analysis. Anal. Chem. 93(44) 2021: 1479214801; https://doi.org/10.1021/acs.analchem.1c03533.

85 Macias LA, et al. Spacer Fidelity Assessments of Guide RNA by Top-Down Mass Spectrometry. ACS Cent. Sci. 9(7) 2023: 14371452; https://doi.org/10.1021/acscentsci.3c00289.

86 Wolk S. Characterization of gRNAs and Ribonucleoproteins for CRISPR Applications [Presentation]. Gene Therapy Analytical Development Europe 2022. Editas Medicine: Cambridge, MA, 2022; https://www.editasmedicine.com/wp-content/uploads/2022/06/Wolk-GTAD-Europe-2022-final-01JUN2022.pdf.

87 Camperi J, et al. Physicochemical and Functional Characterization of Differential CRISPR-Cas9 Ribonucleoprotein Complexes. Anal. Chem. 94(2) 2022: 14321440; https://doi.org/10.1021/acs.analchem.1c04795.

88 Toms HA, et al. Chapter 12. Lentiviral Gene Therapy Vectors: Challenges and Future Directions. Gene Therapy Tools and Potential Applications. Martn-Molina F, Ed. IntechOpen Limited: London, UK, 2013; https://doi.org/10.5772/52534.

89 Elizalde N, Ramrez JC. Lentiviral Vectors: Key Challenges and New Developments. Cell Gene Ther. Ins. 7(6) 2021: 667677; https://doi.org/10.18609/cgti.2021.002.

90 Ferreira MV, Cabral ET, Coroadinha AS. Progress and Perspectives in the Development of Lentiviral Vector Producer Cells. Biotechnol. J. 16(1) 2021: e2000017; https://doi.org/10.1002/biot.202000017.

91 McCarron A, et al. Challenges of Up-Scaling Lentivirus Production and Processing. J. Biotechnol. 240, 2016: 2330; https://doi.org/10.1016/j.jbiotec.2016.10.016.

92 Martnez-Molina E, et al. Large-Scale Production of Lentiviral Vectors: Current Perspectives and Challenges. Pharmaceutics 12(11) 2020: 1051; https://doi.org/10.3390/pharmaceutics12111051.

93 Geraerts M, et al. Comparison of Lentiviral Vector Titration Methods. BMC Biotechnol. 6(34) 2006; https://doi.org/10.1186/1472-6750-6-34.

94 Sastry L, et al. Titering Lentiviral Vectors: Comparison of DNA, RNA and Marker Expression Methods. Gene Ther. 9(17) 2002: 11551162; https://doi.org/10.1038/sj.gt.3301731.

95 Ausubel L, et al. Production of CGMP-Grade Lentiviral Vectors. BioProcess Int. 10(2) 2012: 3243; https://www.bioprocessintl.com/sponsored-content/production-of-cgmp-grade-lentiviral-vectors.

96 Dull T, et al. A Third-Generation Lentivirus Vector with a Conditional Packaging System. J. Virol. 72(11) 1998: 84638471; https://doi.org/10.1128/jvi.72.11.8463-8471.1998.

97 CBER. Guidance for Industry: Testing of Retroviral Vector-Based Human Gene Therapy Products for Replication Competent Retrovirus During Product Manufacture and Patient Follow-Up. US Food and Drug Administration: Silver Spring, MD, 2020; https://www.fda.gov/media/113790/download.

Corresponding author Brent Morse is a principal consultant, and Alicja Fiedorowicz is an analytical consultant in cell and gene therapy, both at Dark Horse Consulting Group near Boston, MA; [emailprotected].

Continue reading here:
Analytical Considerations for Gene-Modified Hematopoietic Stem and Progenitor Cell Therapies: Part 2 Starting ... - BioProcess Insider

Volodymyr Usov is a technology entrepreneur from Ukraine who served as a chairman of the State Space Agency of Ukraine in 2020 and 2021. He is a co-founder of Kurs Orbital, a start-up developing an autonomous rendezvous and docking system for future in-orbit servicing missions.

SpaceX CEO Elon Musk is realistic when it comes to the dangers of settling humans on mars.

"If an arduous and dangerous journey where you might not come back alive, but it's a glorious adventure, sounds appealing, Mars is the place," Musk said in 2021. That's the ad for Mars! A bunch of people will probably die in the beginning."

As we witness substantial advancements with SpaceX's Starship, despite numerous explosions during the tests an acceptable risk for an innovative spacecraft pushing boundaries the prospect of its successful first orbital launch is becoming an increasingly tangible reality. Consequently, Elon Musk's vision of Mars missions and the establishment of initial settlements begins to transcend the realm of dreams and venture into the sphere of achievable objectives.

Hence, this progress invites us to delve deeper into understanding the most significant challenges that lie ahead. These challenges stretch beyond the boundaries of rocket technology, impacting our biology and fundamentally questioning our identity as a species.

Related: Watch SpaceX launch a Starship to Mars in this gorgeous new animation

On Mars, a hostile and radiation-soaked, lifeless world, merely arriving and landing alive is tough for humans, let alone the colossal challenge of survival. It resembles more a celestial tomb than a garden for life. Some thinkers are beginning to ponder, though: Could we craft a new iteration of humanity, genetically sculpted to endure the harsh reality of space travel? In other words, could astronauts be transformed at a genetic level to prepare them for another world?

To clarify, no one is currently nurturing a genetically enhanced astronaut in a lab. At least, not to my knowledge. Yet, ideas once confined to the realm of science fiction are materializing into tangible concepts. We know that radiation, a potent hazard in space, can induce cancer and other serious maladies. However, Chinese scientists have already made strides in genetically modifying human embryonic stem cells to show supernatural resistance against radiation.

As space is flooded with energetic particles that can damage DNA, scientists have proposed the addition of extra copies of p53, a gene known as the "protector of the genome" due to its role in cancer prevention. Elephants, with their surplus copies of p53, rarely succumb to cancer. Perhaps our future astronauts should follow suit.

Demonstrating the feasibility of such a concept, first gene-editing experiments aboard the ISS has proven the effectiveness of CRISPR technology in space. This offers a promising sign of potential breakthroughs to come. There's no consortium focused on genetic engineering for astronauts yet, but perhaps it's time to consider establishing one.

In the quest to shield astronauts, we may also stumble upon opportunities for "enhancement". Currently, the notion of gene editing for intellect enhancement or perfect vision is fiercely resisted. Yet, if we're honest, NASA already selects individuals based on similar criteria. Out of 12 000 applicants, only 10 were selected into its astronaut class in 2021 to train for future missions. You may be familiar with the movie "Gattaca", in which only genetically superior individuals were permitted to journey to Titan, while those deemed genetically inferior looked on enviously. Like much of compelling science fiction, this 1997 film isn't far removed from reality.

When contemplating survival in space, the genetic concept of "fitness" becomes critical. It refers not to physical prowess but to an organism's ability to thrive and reproduce within a given environment.

In space or on Mars, human fitness is perilously low. Consider an astronaut encapsulated within a suit, the environmental conditions meticulously controlled to keep the wearer alive. But the suit exists solely to mimic the terrestrial environment for which our genes have adapted through millions of years of evolution.

Scientists have begun identifying genes that might enhance our survivability. Are you fortunate enough to possess the EPAS1 variant common in Tibetans, which allows for better survival at lower oxygen levels? How about the natural mutation that leads to lean, robust muscles, potentially offsetting the atrophy of space travel? Some individuals even carry a DNA variant associated with excellent problem-solving skills and low anxiety, a trait that would have greatly assisted Matt Damon's character in his survival efforts on Mars in the film "The Martian".

The odds of possessing all these beneficial mutations are astronomically low. This is why we might consider actively incorporating these traits, potentially using next-generation gene editing technology. George Church, a luminary in the field of genetics at Harvard Medical School, has already compiled a list of rare protective gene variants relevant to an extraterrestrial environment including increased resistance to pain, virus resistance, reduced risk of diabetes, cancer and Alzheimer's and even low odor production.

Church posits that we are already transhumanist, having evolved to the point where our ancestors would hardly recognize us. And his argument carries considerable weight. In our quest to explore the cosmos, we confront not just the challenges of spacecraft engineering, but also the equally complex arena of biological engineering. To survive the harsh environment of space, we must not just adapt but evolve, and do so rapidly. We cannot solely depend on natural selection, a slow process demanding large populations and millions of years of evolution in favorable climate those are luxuries we won't have in space.

In a study published in the International Journal of Astrobiology, Matthew R. Edwards explored several cosmic habitation strategies. The conventional model of space colonies, Mars serving as an archetypal example, was matched against the rather unorthodox concept of Embryo Space Colonization (ESC). This audacious model posits the transmission of human embryos to extraterrestrial colonies, where their development into adulthood would be overseen by a fusion of ectogenesis and robotics.

Intriguingly, the analysis suggests that this futuristic paradigm holds greater promise for securing our species' long-term survival in the cosmos compared to conventional colonial establishments.

Traditional space colonies are encumbered by an array of significant obstacles. Among the challenges we face on Mars is the scarcity of CO2 and the unfamiliarity of Mars' gravity, which is approximately 38% that of Earth's. These conditions are complicated by an inhospitable environment saturated with potentially lethal radiation. It makes such colonies less than optimal platforms for humanity's aspiration to venture beyond our home planet, and even more challenging for fostering a new generation within the vast expanse of our solar system. It appears highly unlikely that we could rely on our Earth-familiar methods of natural procreation within such severe extraterrestrial conditions.

Recently, we've witnessed noteworthy advancements in the early prototypes of ectogenesis a process that enables fetal development entirely outside the human body. This concept was first proposed a century ago by the renowned Cambridge biologist, J.B.S. Haldane. The futuristic reproductive science he envisioned, albeit optimistic, was frighteningly reimagined into a dystopian landscape in the initial chapters of Huxley's "Brave New World." Today, a reassessment of this perspective seems necessary, considering the integral role it could play in our long-term survival in space.

Currently, several international research groups are breaking new ground with fetal life-support systems. These promising inventions could potentially nurture the life of extremely premature babies in an environment akin to a womb. Research teams from the US, Australia, and Japan have engineered innovative artificial wombs, such as the Biobag and the EVE platform. These have achieved some success with highly premature lamb fetuses. Concurrently, a Dutch team is exploring a perinatal life support (PLS) system using advanced simulation technology.

Significant strides have been made in imitating the conditions of the womb during late-stage pregnancy. However, our understanding of the earliest weeks remains limited. This is due to the immense difficulty in observing in-womb events, coupled with past restrictions on research involving human embryo development outside the womb beyond 14 days. These regulations are now easing, allowing case-by-case considerations. This paves the way for the progression of artificial womb technology, even though the scientific hurdles in gestating a viable human baby outside the body remain.

In one such instance, scientists at Israel's Weizmann Institute of Science managed to grow mouse embryos ex utero for about 11 to 12 days, slightly over half their gestation period. While these embryos developed organs and limbs, the team continues to grapple with the challenge of extending this process beyond the halfway point.

This is where technology companies like Colossal Biosciences can play a transformative role. Colossal, primarily known for its pioneering work in Mammoth de-extinction and other almost science fiction research, could revolutionize the field of ectogenesis. Colossal's CEO, Ben Lamm, has acknowledged that large-scale de-extinction would necessitate ectogenesis rather than traditional surrogacy. In the interest of social acceptance, he prefers to use the term 'ex utero' rather than 'artificial wombs.'

With its formidable team of top-tier researchers and scientists, led by Lamm's co-founder George Church, Colossal is a strong candidate to actualize full ectogenesis and artificial womb technology. After recently securing $250 million in investment at a $1 billion valuation, the company has the financial resources to match its innovative spirit.

It takes a special kind of genius raising hundreds of millions from VCs to de-extinct Wooly Mammoth and Dodo, and let me tell you, Ben Lamm has that genius in spades. Figures like Elon Musk, Ben Lamm, and George Church have all the potential to redefine our limits. By employing genetic modifications and ectogenesis, they could equip humanity for the unique challenges of the cosmic environment, aiding our transformation into a truly spacefaring civilization. In doing so, we become architects of our own evolution.

Once, the likes of Copernicus and Darwin demoted humanity from the focal point of the universe to a mere product of evolution on an inconsequential planet. But in the light of our advanced understanding, we see that we are more than just another link in the chain of evolution. We are a historical novelty, capable of guiding the path of evolution itself.

In due time, we will extend our civilization into the final frontier, surmounting our evolutionary limitations through technological and biological enhancements. As of now, humanity remains the sole form of intelligence confirmed with certainty. Therefore, our primary goal must be to preserve the existence of this intelligent life in the universe.

Our genome, then, becomes more than just the blueprint for life on Earth. It transforms into the genome of the cosmos, a testament to humanity's adaptability and resilience.

Read more:
When SpaceX's Starship is ready to settle Mars, will we be? (op-ed) - Space.com

PPTP-seq development and validation

PPTP-seq uses plasmid to integrate each CRISPRi-based TF perturbation and each promoter activity reporter into one construct. Each plasmid contains a CRISPRi cassette that constitutively expresses a single guide RNA (sgRNA) to repress a specific TF in the genome19 and a promoter-reporter cassette to measure the activity of a specific promoter under the TF-repressed condition (Fig.1a, b). A self-cleaving ribozyme, RiboJ, was inserted between the promoter and the gfp reporter gene to produce invariant mRNA sequences, thus eliminating the interference of different promoter sequences with gfp mRNA stability20.

a Schematic of a regulatory network. Perturbing regulators and the recorded responses of genes are used to infer regulatory interactions. b Reporter plasmids used to quantify promoter activity under CRISPRi-based regulator perturbation. A native promoter was cloned upstream of the gfp gene, and a sgRNA was inserted downstream of a constitutive promoter. c Massively parallel promoter activity measurements for a combinatory library. A combinatory library of more than 2.5105 sgRNA-promoter pairs was sorted into 16 bins according to their GFP expression levels. The sgRNA and promoter regions in each bin were sequenced to estimate perturbed promoter activity for each sgRNA-promoter pair. d Sorted promoter activities of all promoters. The gray and red dots respectively represent promoter activities in strains with TF-targeting sgRNAs and negative control sgRNAs. The black line represents sorted median promoter activities across all TFKD conditions. The blue lines indicate 2-fold changes from the median activities. a.u. arbitrary units. Source data are provided as a Source Data file.

To profile genome-wide transcriptional responses for all TFs in E. coli, we constructed a combinatorial plasmid library consisting of both a sgRNA library and a promoter library (Fig.1c). The sgRNA library contains 183 TF-targeting sgRNAs that repress every single known TF gene in the E. coli genome (Supplementary Data1), and contains five non-targeting sgRNAs as negative controls. The promoter library contains 1372 native promoters that cover more than 50% of all operons in E. coli21 (Supplementary Data2). The combinatorial plasmid library was transformed into E. coli strain FR-E01, which carries a dCas9 gene in its chromosome. Transformed cells were first grown in minimal glucose medium to a steady state and sorted into 16 bins based on their fluorescence intensity (Supplementary Fig.1a). More than 20 million cells (including all 16 bins) were sorted in each replicate (Supplementary Fig.1b and Supplementary Data3), and their plasmids were sequenced using the NovaSeq S4 XP Platform, generating an average of 420 million reads from each replicate (Supplementary Fig.1c and Supplementary Data3). To estimate promoter activities under each perturbed TF condition, sequencing read counts across the bins were first converted to cell count distribution for each individual variant, followed by fitting into log-normal distribution by maximum-likelihood estimation22,23,24 (Supplementary Fig.2 and Methods).

Measured promoter activities were highly consistent between independent biological replicates performed in different weeks, with replicate correlation ranging between 0.90 and 0.95 (Supplementary Fig.3a). Across three independent replicates, the promoter activities of 201,433 library members (i.e., 201,433 different TF-promoter pairs, 81% of the entire library) passed our quality filters (Supplementary Fig.3b, Methods). For most promoters, the median activity of a promoter across all TFKD conditions was consistent with its activity in negative controls (Fig.1d and Supplementary Fig.4). We found that more than 98% of TF-promoter pairs fell within the two-fold-change boundaries of the median activity, indicating robust promoter activities in most TFKD conditions18,25.

CRISPRi can impair cell growth if essential genes are targeted. Seven TF-targeting sgRNAs (alaS, bluR, dicA, dnaA, iscR, mraZ, and nrdR) had substantially reduced reads (fewer than 10,000 reads per sgRNA compared to an average of 4.8 million reads per sgRNA). Among them, alaS, dicA, and dnaA are essential genes whose deletion led to cell death26,27. CRISPRi polarity28,29 can also lead to the repression of essential genes that are located downstream of a targeting TF within the same operon. This explains the substantially reduced reads for iscR, mraZ, and nrdR.

We further evaluated the CRISPRi repression efficiency using both TFspromoter activity measured from PPTP-seq (Supplementary Fig.5a) and transcript level measured from RT-qPCR (Supplementary Fig.5b). The two methods respectively found 95% and 86% of tested TFs showed significant repression (Students t-test P-value<0.05) compared to their corresponding controls containing non-targeting sgRNAs (Supplementary Note1). We further found a clear negative correlation between the degree of CRISPRi repression and TF expression level measured from TFspromoter activity (Supplementary Fig.5c, d). This explains the lack of repression for the small fraction of TFs (e.g., qseB and ttdR).

To further validate the promoter activities measured by PPTP-seq, we randomly selected five promoters, which involve a diverse range of gene functions. We then individually measured their activities in response to CRISPRi repression of nine representative TFs (and one non-targeting sgRNA as a negative control), using a plate-reader-based whole-cell fluorescence assay (Supplementary Fig.6a). Of these 50 sgRNA-promoter pairs, 45 were quantified by PPTP-seq and were highly consistent with individual whole-cell fluorescence measurements (Supplementary Fig.6b, Pearsons r=0.95), confirming the high quality of our pooled measurements. The other five combinations were missing in all three replicates due to their low read counts. This small dataset also contained the regulatory effects of five known direct interactions and one indirect interaction in RegulonDB1 (Supplementary Fig.6c).

We also compared our promoter activity measurements to previously published datasets from other independent experiments. Promoter activities measured from PPTP-seq (using the negative control strains) correlated with transcript levels measured from RNA-seq30 and promoter activities individually measured using flow cytometry31 (Supplementary Fig.7ac, Pearsons r=0.68 and 0.74, respectively). Additionally, fold change in promoter activity upon TFKD measured from PPTP-seq is also qualitatively consistent with that measured from EcoMAC microarray32 for a few known regulatory interactions in RegulonDB1 (Pearsons r=0.51, Supplementary Fig.7d).

We quantified promoter activity changes by TFKD relative to negative controls (Supplementary Fig.4) and modeled the replicated data as log-normal distributed to determine statistical significance. From the 201,433 measured promoter activities, single TFKDs led to upregulation in 3720 TF-promoter pairs and downregulation in 338 pairs (>1.7-fold in promoter activity, q<0.01; Fig.2a) in minimal glucose medium. Most TFs regulate fewer than ten promoters, while a few TFs affect more than 100 promoters (Fig.2b). We also found promoters that are regulated by multiple activators (leading to downregulation by TFKD in Fig.2c) are much less abundant than those regulated by multiple repressors (leading to upregulation in Fig.2c). The most common regulatory effect on a regulated promoter observed in PPTP-seq was single regulation by a single activator or a single repressor (30%, Fig.2c and Supplementary Fig.4), which was consistent with previous datasets measured using other methods1,14.

a Promoter activity changes by TFKD. Dashed lines indicate cutoffs for statistically significant (q<0.01) and substantial (>1.7-fold change) effects. Each dot represents a TF-promoter pair. Upregulation and downregulation by TFKD are shown in red and blue, respectively. A few known interacting TF-promoter pairs are labeled. b Histogram of the number of regulated promoters per TF. Inset in (b) shows histograms over a smaller range. c Histogram of the number of regulating TFs per promoter. d Fractions of constant promoters and variable promoters in each COG category. All COG categories of genes in an operon controlled by a promoter are assigned to the promoter. The dashed line indicates the average fraction of constant promoters over all COG categories. Statistical significance is determined by one-sided Fishers exact test. **P<0.01. Source data are provided as a Source Data file.

Collectively, we identified 936 (71% of 1323 measured promoters) variable promoters with significant activity change under at least one TFKD condition (Supplementary Note2), and the other 29% of the promoters were consideredas constant promoters. Clusters of Orthologous Genes (COG) analysis33 of all downstream genes of these promoters indicated that genes expressed by variable promoters are enriched in the COG class of Carbohydrate transport and metabolism (P=4.4103) (Fig.2d), specifically KEGG pathways in galactose metabolism (eco00052), pentose and glucuronate interconversions (eco00040), starch and sucrose metabolism (eco00500), and amino sugar and nucleotide sugar metabolism (eco00520). Variable promoters also control genes in flagellar and pilus (Supplementary Data4). The results suggested that these functions or activities are more readily subject to regulation under different condition changes. Genes expressed by constant promoters are enriched in inorganic ion transport and metabolism (P=2.6 103), specifically sulfur metabolism (eco00920), ion transport (GO:0006811), and iron ion homeostasis (GO:0055072) (Supplementary Data4), suggesting that these genes play housekeeping roles (Fig.2d).

We systematically investigated whether a TFs promoter can be affected by itself or other TFs. A perturbation-response network between TFs was constructed, where activation and repression represent down- and upregulation by CRISPRi knockdown of an upstream TF, respectively (Fig.3a). In minimal glucose medium, a total of 26 activations and 339 repressions were observed between 126 TFs (Supplementary Data5). Within this dataset, no mutual regulation or repressilators of three or more TFs were observed, likely due to low expression or missing allosteric regulation for some TFs when cells are growing in minimal glucose medium (Supplementary Note3).

a Perturbation-response network of TFs constructed using PPTP-seq data in minimal glucose medium. b Autoregulation of TFs identified by PPTP-seq in minimal glucose medium. Promoter activity fold changes upon the knockdown of TF controlled by the promoter. TF gene names marked in red were selected for validation. Source data are provided in Supplementary Data5.

We then examined TF autoregulatory responses, which have been challenging to study using other methods due to the coupling between perturbation and readout. We identified 12 autoregulated TFs with strong perturbation effects (>1.7-fold in promoter activity, q<0.01) in minimal glucose medium, including two autoregulatory interactions, PgrR and ComR, not present in RegulonDB (Fig.3b). Meanwhile, several previously identified autoregulated TFs (e.g., PhoB, Fur, LldR, etc.) showed only weak perturbation effects (i.e., less than 30% promoter activity change) under our growth conditions in minimal glucose medium. To further validate these findings, we selected seven TF genes and measured their promoter activities across a wide range of TF concentrations using a tunable E. coli TF library34, in which each endogenous TF is replaced by an inducible TF-mCherry fusion (Supplementary Fig.8). Both pgrR and comR promoters showed higher activity at lower TF levels, confirming their negative autoregulation. PgrR autoregulation is consistent with the identified PgrR binding site on its promoter region35. Except for ZraR, four out of five previously identified autoregulated TFs displayed negligible promoter activity changes over a wide TF level range. Thus, the results from the tunable TF library were mostly consistent with PPTP-seq. Our results also suggest that some previously identified TFs lack autoregulatory response when cells are growing in minimal glucose medium and may occur under other growth conditions36,37,38,39, so the interpretation of TF regulation should consider the condition dependency.

PPTP-seq data also allows us to systematically examine gene regulation on complex metabolic pathways. As an example, we selected the one-carbon metabolism (OCM), in which transcriptional regulation was not well characterized in bacteria. OCM is tightly associated with the synthesis of nucleotides, amino acids, and two essential cofactorstetrahydrofolate (THF) and Sadenosylmethionine (SAM), and it plays important roles in cell survival and growth. However, due to the presence of multiple metabolic cycles and interconnected pathway structures, dissecting the regulatory function of OCM remains challenging.

We identified 28 TF genes that can affect at least one promoter in OCM (Supplementary Fig.9). A few genes in methionine and SAM biosynthesis, such as metA, metE, and metK, were observed to be upregulated by metJ knockdown, recapitulating the known feedback control of SAM biosynthesis via MetJ5,40 (Fig.4a). Additionally, we found that metA, metE, and metK were also regulated by other TFs, but in distinct patterns (Fig.4b). For example, metE was found to be activated only by metJ knockdown, while metK was upregulated by knockdown of ten different TFs. This finding is intuitively surprising because MetE and MetK catalyze two consecutive reactions in the methionine cycle, and enzymes from the same pathway are often co-regulated41. The different regulations on metE and metK thus indicate that enzymes catalyzing consecutive steps can have distinct cellular functions: MetE synthesizes methionine for protein synthesis, and MetK produces SAM as a cofactor for metabolic reactions (Fig.4a).

a Promoter activity changes in response to metR and metJ knockdown by CRISPRi. Hcy and SAM control the activity of MetR and MetJ, respectively. NA not applicable, KD knockdown, GTP Guanosine-5-triphosphate, DHPPP 6-hydroxymethyl-7,8-dihydropterin pyrophosphate, PABA para-aminobenzoic acid, DHP dihydropteroate, DHF dihydrofolate, THF tetrahydrofolate, dUMP deoxyuridine monophosphate, dTMP deoxythymidine monophosphate, Met L-methionine, fMet N-formylmethionine, Hcy L-homocysteine, SAM S-adenosylmethionine, SAH S-adenosylhomocysteine, Rib-Hcy S-ribosyl-L-homocysteine. b TF-dependent promoter activity changes for metA, metE, and metK. Each row represents a promoter, and each column stands for a TFKD condition. c Validation of MetR targets. Promoter activities were measured in a metR knockdown strain and, as a control, in a wild-type E. coli strain. Data are presented as meansSD of three replicates from different days. a.u. arbitrary units. Source data are provided as a Source Data file.

The PPTP-seq dataset also revealed the regulatory functions of MetR, previously known only as a regulator of methionine biosynthesis. We found that metR knockdown affected multiple genes in the folate cycle and folate biosynthesis (e.g., metF, thyA, and folE; Fig.4a), not present in RegulonDB1. Previous DAP-seq binding analysis using purified TFs and genomic DNA fragments identified MetR binding sites at metF and folE promoters42, but the in vivo regulatory responses have never been tested. We further verified these regulatory responses using a MetR knockdown strain from the tunable TF library34 (Fig.4c). These findings allow us to discover metabolic feedback control mechanisms in E. coli OCM under homocysteine-starved conditions because MetR binding to DNA requires homocysteine activation43. When homocysteine is limited, cells cannot produce sufficient methionine for translation initiation and elongation. To quickly rescue the cells from their methionine-limited state, MetR-repression of metF must be alleviated, increasing the amount of 5-methyl-THF and preparing for rapid methionine synthesis when the homocysteine level is sufficiently restored. Meanwhile, upregulated metF and thyA by MetR also increase 5,10-methylene THF consumption, which simultaneously reduces 10-formyl-THF due to reversible reactions between these THF species (Fig.4a). Low 10-formyl-THF and methionine can further result in the insufficient formation of initiator tRNA to slow down translation. Additionally, we found that MetR activates folE, whose enzyme product catalyzes the first step in folate biosynthesis (Fig.4a). Thus, homocysteine limitation can also repress folE, thereby decreasing folate biosynthesis. Taken together, these phenomena suggest that MetR helps to block protein translation initiation and folate synthesis in response to low homocysteine and accumulates 5-methyl THF to prepare for rapid methionine biosynthesis once homocysteine is available.

Our genome-wide promoter activity measurements from perturbed TF levels can provide information that complements TF-promoter binding datasets from ChIP-seq, ChIP-exo, DAP-seq, gSELEX, and curated TF binding sites (TFBSs) in RegulonDB1,42,44,45, yielding knowledge about direct and functional TF-promoter interactions. In total, out of the 4058 regulatory responses identified by PPTP-seq in minimal glucose medium, 225 have binding evidence from DAP-seq, and an additional 256 have binding evidence from other binding datasets, altogether representing 12% (481/4058) of the PPTP-seq identified responses (Fig.5a, b, Supplementary Data6). For 127 TFs with binding site information, on average, 23% of regulated promoters per TF were presumably direct targets (Fig.5c). For the rest 56 TFs, their TFBSs were either not in our promoter library or not identified yet. Among the 481 regulatory responses with binding evidence, only 78 of them were found in the TF-operon network in RegulonDB, and the rest 403 TF-promoter responses may contribute to regulatory interactionsnot present in RegulonDB in minimal glucose medium (Supplementary Table1).

a Comparison of TF perturbation-response results from PPTP-seq and TF binding results. b Fraction of TF-promoter pairs that have binding evidence. c Distribution of fraction of regulated promoters with corresponding TFBS for each TF. dh Factors that may affect whether a potentially bound TF on a promoter affects the promoter activity. For each TF-promoter binding interaction, the binding site location in DAP-seq (d), TF concentration measured by Ribo-seq (e), TF concentration measured by mass spectrometry (f), relative binding strength per TF measured by DAP-seq (g), relative binding strength per TF measured by gSELEX (h), and relative binding strength per promoter measured by DAP-seq (i) were considered. The violin plot shows the distribution of data, the central dot in the box represents the median, the box bounds represent the 25th and 75th percentiles, and whiskers represent the minima to maxima values. The number of TFBSs is indicated below. BenjaminiHochberg adjusted P-values were calculated by the Wilcoxon rank sum test. Source data are provided in Supplementary Data6.

In general, PPTP-seq results and the binding datasets have a small overlap in TF-promoter interaction pairs (Fig.5a), which is consistent with the low overlaps between similar comparisons on specific TFs (GadX, GadW, Fur, and SoxS) in E. coli36,46,47 and between eukaryotic transcriptional response and TF binding datasets3,48. This can be caused by low TF expression levels, low TF activity (affected by other molecules), and/or complex regulatory patterns. We individually examined two promoters that have multiple different TF binding sites (Supplementary Note 4 and Supplementary Fig.10). We found the lack of response can be explained by the context-dependent transcriptional regulation49regulatory function of one TF affected by other TFs bound on the same promoter. Further, we found that deactivating the regulating TF can lead the promoter to respond to previously non-regulatory TFs (Supplementary Note4 and Supplementary Fig.10h, i). These observations indicate that TF-promoter binding is not sufficient for response, and E. coli uses layered control to achieve complex logic for gene expression. In RegulonDB, 48% of regulated promoters have more than one functional TF binding site (Supplementary Fig.11), suggesting that such context-dependent transcriptional regulation can be ubiquitous in E. coli.

We sought to explore what general features determine whether a potentially bound TF can regulate promoter activity under our experimental condition (i.e., growing in minimal glucose medium). For each TF binding site, we focused on the binding location, TF concentration, and binding strength. We found that binding sites from both regulating and non-regulating TFs were centered around the transcription start site (TSS) of a promoter50 (Fig.5d) and that regulating TFs had a significantly higher concentration in cells over non-regulating TFs (Fig.5e, f). Additionally, previous biophysical models indicate that TF-DNA binding energy can predict fold changes in promoter response16,51,52,53. We first hypothesized that when a TF has binding sites at multiple promoters, it tends to regulate its targets with the strongest binding strength. To test this hypothesis, we normalized the binding strength of each TF-promoter pair to the maximum binding strength for that TF (called relative binding strength per TF). On average, the relative binding strength per TF was slightly weaker for regulatory TF-promoter pairs than for non-regulatory TF-promoter pairs (Fig.5g, h). This unexpected result suggests that TFs do not necessarily regulate their most tightly associated promoters. We then considered the affinity of all TFs binding to the same promoter and normalized the binding strength of each TF-promoter pair to the maximal strength of the most tightly associated TF for each promoter (called relative binding strength per promoter) (Fig.5i). Results indicate that for each promoter, TFs with stronger binding are more likely to cause promoter activity change. Taking these findings together, the relative binding strengths of TFs on a promoter are a major determinant of promoter response.

To explore genome-scale regulatory networks at conditions other than minimal glucose medium, we further performed PPTP-seq experiments for cells grown in LB and minimal glycerol media. A total of 5279 and 3810 TF-promoter responses were identified in LB and minimal glycerol media, respectively (Supplementary Fig.12). The larger number of responses seen in LB was partially caused by high TF activity of a few TFs that have specific effectors in rich media (Supplementary Table2). Comparing these datasets with that collected from minimal glucose medium, 867 TF-promoter pairs appeared in all three conditions, with 1901, 2274, and 3495 pairs appearing only in one condition, suggesting TF-promoter responses are highly condition-specific (Fig.6a). The upregulated TF-promoter pairs by TFKD (TF repression) have more overlaps among these three conditions than downregulated pairs (TF activation, Fig.6a), suggesting that TF activation is more sensitive to growth conditions (e.g., affected by allosteric regulation) than TF repression. We examined a few individual TFs with known targets (Supplementary Data7) that have distinct regulatory responses in different conditions (Fig.6b). For example, repression of lacZ promoter by CRP was not detected in minimal glucose medium due to low cAMP concentration54, but was observed in LB medium. Similarly, activation of the maltose transporter malK by MalT was observed in LB medium but not in the minimal glucose medium, because expression of malT requires CRP activation55. On the other hand, activation of metE by MetR was observed in minimal glucose and glycerol media but not in LB medium. This is likely caused by repression of metE by MetJ at high SAM concentration56. Our data show that many regulatory responses are condition-dependent (Fig.6b) and highlight that growth condition needs to be specified when describing the regulatory network.

a Comparison of TF perturbation-response results from PPTP-seq at different growth conditions. b Known TF-promoter interactions from RegulonDB showed different regulation under different growth media. Source data are provided as a Source Data file.

Here is the original post:
Genome-wide promoter responses to CRISPR perturbations of ... - Nature.com

September 11, 2023

Faculty members in the Department of Psychology, a unit within The College of Liberal Arts and Sciences at Arizona State University, received exceptional research awards and scholarly accolades leading up to the new semester.

The diversity of projects and awards announced this summer reflects the breadth of expertise in ASUs psychology department. Faculty members are organized into six specializations: behavioral neuroscience and comparative psychology; clinical psychology; cognitive science; developmental psychology; quantitative research methods; and social psychology. Professor Tamera Schneider, chair of the department, says shes impressed with the quality and breadth of research, as well as the collaborative spirit in the department.

Were committed to developing foundations and deploying solutions for healthy minds, bodies and societies," she said. "Im extremely proud of the innovative work were doing. From cells to society, our researchers are improving lives and communities."

Take a closer look at what psychology faculty will be working on this fall:

Athena Aktipis

Aktipis, an associate professor and director of The Cooperation Lab, has been awarded $1.5 million by the National Science Foundation to tackle the growing gap in societys ability to manage risk, especially those stemming from ecological changes and natural disasters.

Rare events like floods and droughts are becoming more common, and misinformation about hazards, risks and how to manage these events is being exasperatingly spread through the internet, explained Aktipis. As the principal investigator on the grant-funded project, Aktipis and her team will employ gamification and narrative storytelling to benefit vulnerable communities and risk managers by developing effective strategies and outreach initiatives.

Under this grant, three app-based video games will be designed, including The Survival Game, in which players manage herds of cows, fostering cooperation for survival. Aktipis and her colleagues originally developed this game concept for the Exploratorium, a science museum in San Francisco. The goal is for participants to learn more about managing risk through need-based sharing and other risk management strategies.

Aktipis a cooperation theorist, social psychologist, theoretical evolutionary biologist and cancer biologist believes teamwork and cooperation are some of the most powerful forces in the world.

This work will reach diverse segments of society from low-income communities struggling to deal with disasters to water managers in the desert Southwest trying to increase the resilience of the water supply," she said. "Those who will be most positively impacted are those who are most vulnerable, including communities in regions with high risk of natural hazards.

To learn more from Aktipis, tune into ASU Learning Sparks, where ASU faculty transform complex ideas into easily digestible educational experiences.

Olive, professor and head of the Addiction Neuroscience Lab at ASU, examines how abused drugs affect the brain on a neurobiological level. He was granted a research fund of $1.7 million from the National Institute on Alcohol Abuse and Alcoholism (NIAAA) to investigate the neural mechanisms behind binge drinking.

Olives prior research discovered that binge alcohol consumption activates specific endorphin-producing neurons in the hypothalamus, a brain region linked to behavior regulations. Specifically, the arcuate nucleus, rich in endorphin-producing neurons, forms connections with the amygdala, which controls emotions. The NIAAA funded study will expand on this research, investigating brain circuits associated with excessive drinking.

Characterized by intricate connections between various nerve cells and the involvement of different types of chemical signals, brain circuits are not limited to neurons alone. Non-neuron cells also participate in coordinated activity across brain regions. Olive explained this study will determine exactly what subtypes of endorphin neurons and circuits in the brain are sensitive to binge drinking, leading to more effective addiction treatments and improved outcomes for those facing addiction-related challenges.

Our hope is to identify specific circuits in the brain whose primary chemical messengers endorphins regulate binge drinking and how these circuits go awry when someone binge drinks repeatedly to the point of self-harm, Olive said. With that knowledge in hand, hopefully newer neuromodulation technologies that allow for precise retuning of specific brain circuits can be used as intervention strategies for individuals struggling with alcohol dependence and uncontrollable episodes of binge drinking.

Sandler, a research professor and Regents Professor emeritus at the Research and Education Advancing Childrens Health (REACH) Institute in ASUs Department of Psychology, has been awarded a $925,000 research grant from The New York Life Foundation. The grant aims to evaluate the effectiveness of a digital program designed to aid caregivers of children who have experienced the death of a parent and to facilitate its widespread dissemination.

His prior work on the Family Bereavement Program, which was funded by the National Institute of Mental Health, involved a randomized trial that demonstrated significant impact in preventing long-term mental health issues of children who had experienced the death of a parent. The program reduced the incidence of major depression in bereaved youth, even fifteen years after, and demonstrated significant long-term benefits for the surviving bereaved, including decreased prevalence of prolonged grief-related distress six years down the line.The New York Life Foundation supported Sandler and his team in translating these experimental results into a service that can be easily provided by community-based service providers. They have trained numerous individuals to deliver the caregiver component of the Resilient Parenting for Bereaved Families program. An evaluation has confirmed its positive impact in strengthening caregiver-child relationships, alleviating caregiver-complicated grief and reducing child behavior problems.

Now, The New York Life Foundation is assisting Sandler and his team in digitizing the program into the Online Resilient Parenting for Bereaved Families Program (eRPBF) to reach a wider population of caregivers of children who have experienced the death of a parent. Over the course of three years, the new grant enables Sandler and his team to partner with community agencies and professionals that work with bereaved families to evaluate and disseminate the digital program. The grant will also aid in developing cultural adaptations of the program that make it fully resonant with the life experiences of African American and Latino bereaved families.

Its been both an intellectual challenge and a personal privilege to develop research-based tools that can support caregivers and their families following the death of a parent," Sandler said. "Our challenge now is to make these programs accessible to all families who need them so that they really make a difference in the lives of children.

Shiota, professor and director of the Shiota Psychophysiology Laboratory for Affective Testing (SPLAT) Lab at ASU, has launched not one, but two funding projects totaling over $270,000. Both grant-funded projects address the escalating opioid crisis.

One project, a collaboration with REAL Prevention, refines and evaluates a new technology aimed at reducing deaths by opioid overdose. By teaching community responders to use Naloxone a nasal spray that can rapidly reverse an opioid overdose and using an app called PulsePoint to alert community responders to a possible overdose happening nearby, the Opioid Rapid Response System directs lifesaving measures to people in need until emergency services can arrive. Shiota will help develop the training program and assess effects on community responders knowledge and confidence in administering Naloxone. The project will monitor the overall impact on participating communities as well, in terms of overdose survival rates.

In the second project, Shiota leads a contract between the city of Phoenix and the Substance Use and Addiction Translational Research Network (SATRN) a collective of university researchers, community-based prevention and treatment practitioners, and policymakers across the state of Arizona dedicated to reducing death and distress associated with substance use disorder. Shiota and other SATRN affiliates will advise the city of Phoenix on potential uses for opioid settlement funds, developing and analyzing assessment surveys and recommending training and other initiatives addressing the most pressing needs.

City of Phoenix residents and employees alike are encountering people struggling with opioid-related problems in their daily lives. Through this partnership, SATRN is helping to capture and understand peoples experiences, and learn what initiatives residents and city staff think would be most helpful, Shiota said. While these two projects differ in many ways, both engage community members in helping to save lives and rely on teamwork and knowledge-sharing to develop solutions.

Anderson, an assistant professor in quantitative psychology, was elected into the Society of Multivariate Experimental Psychology (SMEP). This distinguished assembly of 65 experts champions multivariate quantitative methods application in psychology and allied fields. An individuals SMEP membership spans from the time of election to the age of 65.

It is such an honor to have been elected into SMEP by my quantitative methods colleagues, especially this early in my career. So many of the greats of my field have been members of this organization, and I am humbled to be a new part of such a longstanding research society, Anderson said.

Anderson joined ASU in 2018. She probes research design, statistical methods and metascience, spotlighting practical and rigorous approaches that encompass potent sample size planning, replication remedies, multiplicitys impact on Type 1 error rates and power, and approaches for missing data.

Driven to enhance accessibility, Anderson co-developed open-source software for unbiased sample size planning and recently received the Rising Star Award from the Association for Psychological Science for her pioneering early-career research.

MacKinnon, Regents Professor and director of the Research in Prevention Lab, instructs graduate analysis of variance, mediation analysis and statistical methods in prevention research courses at ASU. This fall, hell further amplify his influence by serving as a McCausland Visiting Scholar at the University of South Carolina (USC). This premier faculty program is reserved for award-winning, impactful researchers who foster interdisciplinary collaboration.

MacKinnons distinguished career encompasses vital roles, including as a founding member and inaugural fellow of the Society for Prevention Research (SPR), as well as serving as a fellow of the Association for Psychological Science and the American Psychological Association's Division 5: Quantitative and Qualitative Methods. He has also previously served as president of the Society for Multivariate Experimental Psychology.

His decades of experience in research and leadership in quantitative methodology offers a unique perspective on the evolution of quantitative psychology and its promising research avenues. As a McCausland Visiting Scholar, MacKinnon will expand on his existing collaborations with USC researchers by delivering guest lectures to students, engaging with faculty like Amanda Fairchild and presenting compelling public seminars.

They have an outstanding group of quantitative and substantive psychologists at USC. The quantitative faculty conduct research in some of the major new directions in this area, MacKinnon said. I am very much looking forward to formal and especially informal discussion of a variety of topics as a McCausland Visiting Scholar.

Read more:
Expanding the toolbox for RNA editing | ASU News - ASU News Now

Yatish Turakhia. Photo by Damon Casarez/ MIT Technology Review

The tools were developed by Turakhia as a postdoctoral researcher with Professors Rusell Corbett-Detig and David Haussler at UC Santa Cruz, and were later improved and expanded at UC San Diego by Turakhias lab members. UShER was also separately packaged into a web interface by Angie Hinrichs, a senior software architect with the UCSC Genome Browser team, which led to its wider adoption.

Our global understanding of how covid spreads would have been severely compromised without Yatishs work, said David Haussler to MIT Technology Review. Haussler is the scientific director of the UCSC Genomics Institute. The product of his algorithm, which nobody else could make, is a global picture of how the virus spread in full genetic detail around the entire globe.

Most recently, Turakhia and a team of two undergraduate researchers at the Jacobs School of Engineering at UC San Diego packaged some of this data into a web interface called RiVeT. This interface provides information on the origin of recombinants genetically different versions of the virus that joined to create hybrid strains discovered through RIPPLES. It also links to other visuals of the same recombinants, such as the Taxonium, to help researchers and curators more easily track the relationship and spread of virus strains. RiVeT makes the data from UShER and RIPPLES more useful and easily accessible, and provides weekly updates on the recombinants discovered through RIPPLES.

While all of the data used in these tools had been based on clinical samples, Turakhias team is now developing ways for their tracking tools to work with wastewater data. He received funding from an Amazon Research Award and the Center for Disease Control (CDC) to apply these genomic surveillance software tools to wastewater monitoring.

For a variety of reasons, people are not doing Covid-19 clinical sequencing at the same rate that we saw even a year ago, said Turakhia. The variants continue to evolve, and most of the evidence thats in circulation is coming from wastewater data. This is much cheaper than clinical data, and can give you a population-level analysis. However, its much harder to do wastewater monitoring. My group is working with other researchers, including at UC San Diego and Scripps Research, to see how we can combine our software mapping and recombinant detection tools to make wastewater surveillance a lot more potent for SARS-CoV-2 and other pathogens going forward.

Turakhia said being included as an Innovator Under 35 was an exciting honor personally, but also a welcomed recognition for this research area.

Its great that MIT Technology Review has recognized the importance of genomic surveillance, he said. Theyve placed it alongside other very relevant topics like climate change and AI, which of course have and deserve a lot of public attention. But I think genomic surveillance is also an important field that will help us combat future pandemics and outbreaks, and save lives as a result.

Read this article:
Electrical Engineer Named MIT Technology Review Innovator Under ... - University of California San Diego

Ensuring the former, and eradicating the latter, is what the current cutting edge of medical genetics promises. Yet we are still as fearful of playing God with biology as we ever were.

This week saw the passing of Ian Wilmut who, with his team at Edinburgh University, created the first mammal cloned from an adult cell (the infamous Dolly the Sheep) in 1996. It reminds us of Scotlands key role in the advance however tentative of genetic engineering.

Many of the scare stories raised by Dolly are often centred on the potential for producing copycat embryos for test and experiment. Pro-lifers (from George W Bush downwards) furiously opposed it.

READ MORE:Dolly the Sheep scientists in coronavirus cell treatment talks

The scientists originally looked to extract stem cells from these embryos. These stem cells could be triggered to grow into transplantable organs, or unique medicines, perfectly tailored to their original human sources. But must stem cells be taken from cloned proto-babies? Cue stramash. Yet this specific scare was eventually invented away.

Shinya Yamanaka gained a Nobel Prize, in 2012, for discovering induced pluripotent stem cells. This was material that could be taken from biological adults thus relieving the need to make embryos.

In 2016, speaking to Scientific American, Yamanaka gave full credit to Wilmut for inspiration. Dolly the Sheep told me reprogramming [of the cells nucleus] is possible even in mammalian cells, and encouraged me to start my own project.

Wilmut was genial, bearded and parka-wearing, described by his biographer Roger Highfield as having the face of a bank clerk. So he was a comforting front man for what remains, even today, the most revolutionary possibility: shaping and designing humans and animals at their genetic and cellular level.

These days, the leading genetics story in town is the continuing activity of He Jiankui (or JK, as he likes to be called), the Chinese researcher who enabled the first-ever genetically-edited humans, with the births of the pseudonymous twins Lulu and Nana in November 2018.

JK was jailed for three years by the Chinese government in 2019, for breaking their national bio-regulations. He has been rendered persona-non-grata by many of his fellow genetics scientists.

At the beginning of this month, as The New Yorkers Dana Goodyear reports, JK is now released and setting up new labs at the Wuchang University of Technology, his title director of the Institute of Genetic Medicine. All this based in believe it or not Wuhan.

But Hes crime, as the New Yorker feature painstakingly shows, was only to have been a few steps ahead of where many scientists in the field want to be. Such reprogramming of life is continuous with Wilmuts discovery not some monstrous break with it.

The crucial biotech tool here is the CRISPR method, for which Jennifer Doudna and Emmanuelle Charpentier won the Nobel Prize for Biology in 2020. CRISPR is a viral DNA found in E.coli. Often compared to a pair of scissors, CRISPR can snip away at parts of a DNA sequence. Doudna and Charpentier created a protein which could help target these blades with considerable precision.

What He did was to use CRISPR to cut out a gene called CCR5 from the DNA sequence of these human embryos. CCR5 is known to increase human receptivity to HIV/Aids (the parents were HIV-positive, a badly-regarded disease in China, and wanted their children to live a life free from that judgement).

JKs own goal, given to him on notepaper by James Watson (the original co-discoverer of DNA with Francis Crick) was posted to the wall of his office. It read Make People Better. Hes ambition was to edit humans genes so you can stop the heritable germline of a condition.

Youdont want to just fix it within someones unhealthy body (or somatically, in the biologists jargon), you want to entirely remove the condition from, and for, future generations.

But does this slide into Making Better People? That is, does it raise the chilling prospect of eugenics, or a biological overclass? It could all too easily, say many of the scientists Goodyear interviews in her feature.

A gene editing expert, Fyodor Urnov, provides the scariest quote. Its all too easy for heritable editing to be used for non-therapeutic modifications, says Urnov (or human enhancement). He gives us three use-case scenarios which we should be very afraid about.

Fear number one: the weaponisation of the military. We know how to make a human being who runs on four hours of sleep I can tell you what mutation to make.

Two: We know what gene to edit to reduce pain sensation. If I were a rogue nation wishing to engineer a next generation of quasi-pain-free special-forces soldiers, I know exactly what to do. Its all published. And three: physical strength. You dont need a large lab operation. You just need the ill-will.

READ MORE:Dolly the Sheep creator Sir Ian Wilmut says 'Noah's Ark' of cloned stem cells could halt extinctions

Shudder. Nevertheless, there is something consistent and admirable about how genetic biologists can terrify themselves into self-regulation. Often well ahead of the laws of governments (or the pitchforks of the people).

When gene-splicing between species was made possible in the 70s, the Asilomar conference of 1976 saw a global collection of scientists gathering even across Cold War lines impose tight and mutually-monitored controls on such experiments. They feared superbugs and other abnormalities.

The CRISPR generation of scientists have already been convening rigorously. Yet the accuracy of these genetic scissors becomes ever greater (though they can currently still leave a messy mosaic of cut and uncut DNA).As they get sharper, the next step for which He Jiankui has become something of a scapegoat is likely to be taken.

One scientist talking to Goodyear describes a moral force-field thats bound to weaken as the science gets better There will come a moment when all the big questions have been answered, and where a doctor is facing a patient.

As with so many of the existential risks we face, such god-like tools require at least a Solomonic wisdom. Are we even remotely capable of that? For example, we should worry that JKs self-justification for his human editing was partly about removing the stigma of HIV from infected Chinese families. That seems back to front. Surely its such social prejudices that should be just as open to re-engineering?

We also have societies where wealthy elites are building a separate and defensible world from the rest of us. The idea their offspring might benefit from a growing menu of human enhancements comes from the bottom of the science-fictional barrel but it could be all too real.

From his new Wuhan fastness, He Jiankui aims to take CRISPRs blades to the gene sequences that give rise to Alzheimers and Duchenne muscular dystrophy (a fatal disease causing unstoppable muscle damage among boys). Should we wish him well? It feels wrong not to grant him some success.

Ian Wilmut was eventually beset himself by Parkinsons. He noted in a 2019 interview: I think that unexpectedly the Dolly experiment has revolutionized the approach to these inherited diseases. I really do genuinely believe that treatments will come along but it may very well be 50 years before the treatment becomes routinely available.

So people like me will probably have died of Parkinsons disease before the new treatments become available. Which is a frustrating thing to think.

Yes, thats how a bank clerk might put it. But perhaps we need more the thoughtful woolly jumper-wearer, than the brash biological entrepreneur for this next stage of Dollys legacy.

Link:
Can we rely on our 'moral force-field' to stop cloning going too far? - The National

The companies are collaborating to apply ShapeTXs AAVid capsid discovery platform and transgene engineering technology in addition to Otsukas expertise in genetic payload design and ophthalmology to develop novel treatment options for eye diseases.

Otsuka Pharmaceutical and ShapeTX announced on Sept.7, 2023 a multi-target collaboration to develop intravitreally delivered adeno-associated viruses (AAVs) for ocular diseases, with the possiblity to add additional targets and tissue types. According to the press release, the companies are collaborating to apply ShapeTXs AAVid capsid discovery platform and transgene engineering technology in addition to Otsukas expertise in genetic payload design and ophthalmology to develop novel treatment options for eye diseases.

The AAVid platform combines throughput screening of billions of unique AAV variants and machine learning to identify novel AAV capsids for direct-to-NHP in vivo selection to maximize clinical translation. Further, according to the release, AAVid capsids are designed for precise target tropism while detuning for off-target biodistribution, which reduces the required dose and associated clinical safety risks. In addition, ShapeTXTX will apply the companys transgene engineering technology to optimize payloads provided by Otsuka for therapeutic levels of gene expression in targeted cell types.

Under the new agreement, ShapeTXTX will receive an initial payment from Otsuka and is eligible to receive development, regulatory, and sales milestone payments that will potentially exceed $1.5 billion in aggregate value. ShapeTXTX is also eligible to receive tiered royalties on future sales of products resulting from the collaboration, according to the press release.

Weve built our AAVid platform on generative AI approaches akin to those behind Midjourney and DALL-E 2 to tackle industry challenges with gene therapy delivery, said Francois Vigneault, PhD, co-founder and chief executive officer of ShapeTXTX, in a press release. By incorporating diffusion models, our platform is designing novel medicines that transcend the boundaries of what is possible experimentally. Our collaboration with Otsuka marks an exciting chapter in our journey as we extend the reach and impact of our technologies to help as many patients as possible.

Source: ShapeTX

See the original post here:
Otsuka Collaborates with ShapeTX for Development of AAV Gene ... - Pharmaceutical Technology Magazine

Almost 35 years have passed since Michel Sadelain last sat at a microscope in a University of Alberta lab searching for ways to maybe one day harness the power of the immune system to fight cancer, but it was as clear then as it is now that greatness was just on the horizon.

There are certain students you pick out and say, That ones going to go a long way, says Lorne Tyrrell, a longtime U of A Distinguished Professor, member of the Canadian Medical Hall of Fame, GSK Chair in Virology and leading hepatitis B researcher.

He was a brilliant young man. I always thought he was going to win some major prizes.

Today, Sadelain fulfilled that decades-old prophecy upon being awarded the 2024Breakthrough Prize in Life Sciences in recognition of his discovery of cancer-fighting immunotherapy based on the genetic engineering of a patients own T cells.

I wanted to share some of this recognition with the U of A, as my time there greatly contributed to shaping my relationship to science, stimulating my curiosity and instilling rigour but without stifling imagination, says Sadelain.

An immunologist and director of the Center for Cell Engineering at Memorial Sloan Kettering Cancer Center in New York, Sadelain demonstrated that T cells a type of white blood cell that helps your immune system can be engineered to acquire the ability to recognize and destroy cancer cells.

These refurbished T cells, which he refers to as a living drug, are made by extracting a cancer patients T cells, inserting synthetic antigen receptors, which he named chimeric antigen receptors (CARs), and then reinfusing the cells.

He really was the inventor of the CAR T cell, says Tyrrell.

Sadelain, who hails from France, received an MD at the University of Paris in 1984 before attending the U of A, where he worked under renowned immunology professor Tom Wegmann and his vaunted Department of Immunology.

Drs. Wegmann, Tyrrell, Singh and Green have remained mentors and role models ever since, adds Sadelain.

After earning his PhD in immunology at the U of A in 1989, he left Edmonton to train as a postdoctoral fellow at the Massachusetts Institute of Technology in Cambridge, Mass., where he began his research on genetic engineering of immune cells.

In 1994, Sadelain went to New York, where he established programs on human hematopoietic stem cell and T cell engineering.

As he was finishing his PhD, he had the idea that maybe he could make the immune system fight cancer, says Tyrrell. And he really did believe thats what he could do.

In 2003, Sadelains lab identified a protein-coding gene, CD19, as a target for CAR therapy in mice, and was the first to report on the effectiveness of such treatment in adults with relapsed, lymphoblastic leukemia. The treatment received FDA breakthrough designation in 2014 and full approval in 2017.

Sadelains work retooling the immune system is not only being employed in the fight against leukemia, lymphomas and a number of other cancers, including solid tumours, but is also being used to treat severe hemoglobinopathies, including sickle cell disease.

And while his life and work are centred in New York, the U of A and Edmonton are still a major focus of his life. His sister, brother and mother all have degrees from the U of A, with the latter two still calling Edmonton home. As well, Sadelain still advises on the work of Michael Chu, professor of oncology in the Faculty of Medicine & Dentistry, who is leading a project to manufacture and test locally produced CAR T cells for treating leukemia and lymphoma.

Michel will win more prizes. Its not uncommon that people who win the Breakthrough Prizes often make a trip to Stockholm to accept a Nobel Prize, says Tyrrell.

This award reflects very well on the education and mentorship he received here.

At $3 million for each recipient, the Breakthrough Prizes are the richest awards in recognition of scientific advances. The annual prizes are given in mathematics, fundamental physics and life sciences, and are sponsored by a host of tech entrepreneurs including Mark Zuckerberg and Priscilla Chan of facebook and Sergey Brin, co-founder of Google.

Sadelain shares the award with fellow immunologist Carl June, a University of Pennsylvania researcher who developed and commercialized tisagenlecleucel, the first FDA-approved gene therapy for use in patients with B-cell acute lymphoblastic leukemia.

Fredrick Van Goor, who earned his PhD in biological sciences from the U of A in 1996, also received the Breakthrough Prize in Life Sciences for developingthe first effective medications to treat the underlying cause of cystic fibrosis. In combination, the medications are effective for more than 90 per cent of patients,greatly improving the length and quality of their lives. Van Goor shares the prize with co-discoverers Sabine Hadida and Paul Negulescu.

See original here:
Cancer discovery earns U of A grad the Breakthrough Prize - University of Alberta

Last week, Israeli scientists announced that they had created a model human embryo without using sperm or eggs; are we heading for a future where procreation is an entirely technological, not biological, phenomenon? Genetics and reproduction specialist Dr Gne Taylor explains what this means for the future of human reproduction.

Brave New World-like dystopia, transhumanist pipedream, or feminist paradise? Dr Gne Taylor will be hosting a panel with Anders Sandberg, Mary Harrington and Kristen Godhsee to debate the future of the artificial womb at HowTheLightGetsIn Festival in London, 23rd and 24th September. Check out the festival programme and incredible line-up of speakers here.

It was recently reported that researchers have created models of human embryos out of stem cells in a lab environment, without the use of sperm or eggs, grown outside the womb. Can you explain what exactly this means? Is this a biological entity that could grow further, and go on to be become a human?

A few years ago, scientists found to their surprise that, when under the right conditions, mouse stem cells within the lab can self-organise into structures akin to those seen during early mouse development. This caused much excitement as people wondered whether it would be possible to do the same with human stem cells. This very recent news reported that when scientists mixed four different types of human stem cells together, they organise themselves into these structures that look recognisably like human embryos at an early stage of development complete with cells that would form a placenta etc.

At present, these human stem cell-based embryo models develop at rates slightly different to human embryos and are inefficient to generate in the lab, indicating that more work is required to perfect their growing conditions. Even further work will be required to see if these models do accurately replicate early human development it is still very early days. Ultimately, until they are tested by growing further it is not possible to say definitively if they could become a human.

It sounds like an amazing achievement, but whats the point in growing artificial embryos from stem cells? What are the possible benefits?

Human embryos spend a critical period of their early development hidden away inside the lining of the mothers womb, which makes it hard to study them. Many important biological events occur within the embryo during this time and there is also a high incidence of embryo loss. Therefore, understanding what is happening within human embryos during this period of early embryonic development will help us better understand early pregnancy loss and how birth defects occur.

Currently, scientists rely upon non-human animal models such as mice to try and understand these early stages of development. The hope for this new field of generating embryo models from stem cells is to allow scientists to grow structures that resemble human embryos outside of the human body, therefore allowing them to be studied. As these models use human cells, it is becoming possible for the first time to start really understanding what goes on during this black box of our development.

SUGGESTED VIEWING The Contraception Delusion With Gne Taylor

What are the implications of this development for the internationally recognized ethical limit (the 14-day rule) for growing embryos outside of the womb? Do you expect the ethical guidelines on this to change given this recent development?

The 14-day rule was developed in the 1970s in response to public concerns regarding in vitro fertilisation (IVF). Through innovative and extensive public dialogue and consultation, the Warnock Committee established that human embryos should not be cultured in a lab beyond 14 days of development. This new and growing field of stem cell-based embryo models did not exist when the 14 day rule was established. Therefore, whether or not the 14-day rule applies to these new models comes down to your definition of an embryo. An international group of scientists working within the field have proposed a series of tipping points for when these human embryo models could eventually be afforded similar legal and ethical protection as that of human embryos. Consequently, I do expect that ethical guidelines on this will change, as will the definition of what an embryo is.

Fertility technology and genetic engineering technology such as CRISPR raise numerous ethical questions. Are there new ethical questions that are raised because of this new achievement?

Of course. New innovations always raise new perspectives and questions, and I am certain there will be many. For me personally, one of the most interesting ethical questions that has immediately sprung to mind is: is it more ethical to use animal models or generate enough human embryo models to do meaningful experiments directly in the human context? Of course, adult disease experiments will still need animal models, but these stem cell-based embryo models do present a tantalising opportunity to reduce the number of animals used in research.

___

Our clear sense of what it means to be human is being challenged by these kinds of experiments just as IVF once challenged what it means to have a baby.

___

How might lab grown embryos redefine what it means to be human?

This is at the heart of the matter. Our clear sense of what it means to be human is being challenged by these kinds of experiments just as IVF once challenged what it means to have a baby. Only time will tell which facets are the most important to different cultures and how the diversity of human perspectives and values will be translated into legal and ethical frameworks to regulate these embryo models.

What will the future of human development look like if reproduction no longer requires a sperm, egg or womb? Are we heading for utopia, where our current reproductive problems are all solved, or a dystopia where genetic engineering creates a new underclass?

These new embryo models replicate the very earliest stages of pregnancy not the whole 9 months! So while this is a great opportunity for us to reflect on how we think the tools we have at our disposal should be used to improve lives in the future, it remains to be seen if it is even possible to grow a baby without a womb. Its much too early to be celebrating the advent of a reproductive utopia, sadly!

___

Having knowledge does not mean it must be utilised nor guarantees it will be used.

___

With new technology, like AI and CRISPR, arises the question of whether scientists should be doing something just because they can, because they are capable of pushing new boundaries. Whats your view on this? Should there be no-go areas or moratoriums when it comes to new technology that can alter the future in ways we cant predict?

Personally, I dont see that not knowing something is protection from it. I believe that pushing the boundaries of knowledge and what is possible is the function of science. Also, that knowledge permits us to assess our options, and the courses of action we have available in any given moment, to make the changes we wish to make. However, we all know intuitively that there is a difference between having knowledge and using knowledge Having knowledge does not mean it must be utilised nor guarantees it will be used.

The recent paper: https://www.nature.com/articles/s41586-023-06604-5

Key review of the policy and governance of embryo models: https://www.sciencedirect.com/science/article/pii/S0959437X23000837

Key statement on the ethical framework of embryos: https://www.cell.com/cell/fulltext/S0092-8674(23)00807-3

Read more from the original source:
The Brave New World of synthetic humans | Gne Taylor - IAI

Illustration of a colon cancer cell. [Image: Kateryna Kon / Science Photo Library / Getty Images]

The treatment of malignant tumors has long presented a challenge for cancer researchers. Bacteria-based therapiesin which microbes, often souped-up by genetic engineering or nanotechnology, are put to work as on-site cancer killersshow promise for improving upon conventional methods. But these techniques have drawbacks, including the risk of antibiotic resistanceand the need for complicated procedures that could degrade the bacteria.

Now, researchers in Japan have demonstrated a new approach for enhancing purple photosynthetic bacteria, which they say is an ideal strain for effective cancer phototherapy (Nano Today, doi: 10.1016/j.nantod.2023.101966). Their process, which involves simple chemical functionalization, preserves the innate medicinal qualities of the bacteria while enhancing their ability to fight cancer.

The researchers chose the purple photosynthetic bacteria Rhodopseudomonas palustris(RP) as an optimal candidate for cancer treatment because it is spatiotemporally activatable by near-infrared light and shows strong photothermal conversionthe ability to turn laser light energy into heat, in this case to selectively eliminate cancer cells. This is thanks to its bacteriochlorophyll (BChl) light-harvesting nanocomplexes, which are useful for targeted optical cancer therapies.

RP demonstrated excellent properties, such as near-infrared (NIR) fluorescence, photothermal conversion and low cytotoxicity, explainedlead author Eijiro Miyako, Japan Advanced Institute of Science and Technology (JAIST), in a press release accompanying the paper.Itabsorbs NIR light and produces free radicalsa property that can be utilized to kill cancer cells.

The membranes of photosynthetic bacteria were PEGylated, and fluorescent markers and an anti-PD-L1 antibody were attached to enable tumor-targeting and immunological activation. The engineered bacteria demonstrated effective tumor suppression and immunological responses in a mouse model of colon cancer. [Image: Eijiro Miyako] [Enlarge image]

After selecting their preferred bacteria, the researchers looked to improve them through a series of modifications. First, they attached polyethylene glycol (PEG) derivatives, including one called Biocompatible Anchor for Membrane (BAM), to the bacterial cell walls. Known as PEGylation, this process has a number of benefits, including helping the complex evade host immune response and facilitating attachment of other biomolecules. The team then affixed a fluorescent Alexa488-BSA conjugate to the BAM, which allowed it to be tracked with fluorescence microscopy and used for locating tumors.

Finally, the researchers tacked on an immune checkpoint inhibitor antibody known as anti-PD-L1 using the same BAM method. Cancer cells express a protein called Programmed Cell Death Ligand 1 (PD-L1), which suppresses the hosts immune response and allows cancer cells to evade detection and elimination. Anti-PD-L1 antibodies block PD-L1, thus preventing cancer cells from flying under the immune radar and allowing them to be targeted by the hosts immune system.

To examine the efficacy of the various bacterial complexes, the researchers pitted them against colon cancer in mice in a series of experiments. Tests showed that anti-PD-L1BAMRP, BAMRP and RP inhibited tumor growth when injected in mice with colon cancer. However, all the varieties had an especially dramatic anticancer effect when excited with an NIR laser at 0.7 W for 3 minutes.

During the 30-day follow-up period after the experiment, solid tumors disappeared completely in mice that underwent laser irradiation of injected anti-PD-L1BAMRP, BAMRP, or RP. Laser-induced anti-PD-L1BAMRP was the most effective during the primary treatment stage and also cured tumors faster than the others.

Our findingsrevealed that light-driven functional bacteria demonstrated effective optical and immunological functions in the murine model of colon cancer. Moreover, the NIR fluorescence of the engineered bacterial complexes was used to locate tumors, effectively paving the way for future clinical translation, saysMiyako. We believe that this bacterial technology could be available for clinical trials in 10 years and have positive implications for cancer diagnosis and therapy.

Continue reading here:
Targeting Tumors with Photosynthetic Bacteria - Optics & Photonics News