Gene Therapy Research Research Articles

Newer therapeutic options for inherited retinal diseases: Gene and cell replacement therapy.

Inherited retinal diseases (IRD) are genotypically and phenotypically varied disorders that lead to progressive degeneration of the outer retina and the retinal pigment epithelium (RPE) eventually resulting in severe vision loss. Recent research and developments in gene therapy and cell therapy have shown therapeutic promise in these hitherto incurable diseases. In gene therapy, copies of a healthy gene are introduced into the host cells via a viral vector. Clinical trials for several genes are underway while treatment for RPE65 called voretigene neparvovec, is already approved and commercially available. Cell therapy involves the introduction of stem cells that can replace degenerated cells. These therapies are delivered to the target tissues, namely the photoreceptors (PR) and RPE via subretinal, intravitreal, or suprachoroidal delivery systems. Although there are several limitations to these therapies, they are expected to slow the disease progression and restore some visual functions. Further advances such as gene editing technologies are likely to result in more precise and personalized treatments. Currently, several IRDs such as retinitis pigmentosa, Stargardt disease, Leber congenital amaurosis, choroideremia, achromatopsia, and Usher syndrome are being evaluated for possible gene therapy or cell therapy. It is important to encourage patients to undergo gene testing and maintain a nationwide registry of IRDs. This article provides an overview of the basics of these therapies and their current status.

Gene Therapy for Critical Limb Ischemia: Per Aspera ad Astra.

Peripheral artery diseases remain a serious public health problem. Although there are many traditional methods for their treatment using conservative therapeutic techniques and surgery, gene therapy is an alternative and potentially more effective treatment option especially for "no-option" patients. This review treats the results of many years of research and application of gene therapy as an example of treatment of patients with critical limb ischemia. Data on successful and unsuccessful attempts to use this technology for treating this disease are presented. Trends in changing the paradigm of approaches to therapeutic angiogenesis are noted: from viral vectors to non-viral vectors, from gene transfer to the whole organism to targeted transfer to cells and tissues, from single-gene use to combination of genes; from DNA therapy to RNA therapy, from in vivo therapy to ex vivo therapy.

Recommendations on qPCR/ddPCR assay validation by GCC.

Gene therapy, cell therapy and vaccine research have led to an increased use of qPCR/ddPCR in bioanalytical laboratories. CROs are progressively undertaking the development and validation of qPCR and ddPCR assays. Currently, however, there is limited regulatory guidance for the use of qPCR and a complete lack of any regulatory guidelines for the use of the newer ddPCR to support regulated bioanalysis. Hence, the Global CRO Council in Bioanalysis (GCC) has issued this White Paper to provide; 1) a consensus on the different validation parameters required to support qPCR/ddPCR assays; 2) a harmonized approach to their validation and 3) a consistent development of standard operating procedures (SOPs) for all the bioanalytical laboratories using these techniques.

Characterization of advanced therapies: leveraging advanced analytics to avoid FDA holds

Key assay types needed for successful characterizationCBM’s analytical strategy to address the need Orthogonal approach for early product characterizationNext generation sequencing for product characterizationDana Cipriano has a 20+ year career in biotechnology with leadership roles focused on conceiving and designing processes and programs to drive sustainable growth and profitability. Before joining CBM, Dana was most recently Senior Director of Operations & Strategic Projects for WuXi Advanced Therapies where she directed operations for cell and gene therapy and biologics testing. At WuXi, she created the overall program strategy, drove tactical implementation plans, and monitored key milestones to drive delivery of organizational objectives. She developed a fully integrated program for the development of new corporate strategy centered on driving scalability in cell and gene therapy and biologics testing services. Dana has a Masters in Microbiology from Thomas Jefferson University and a Bachelor’s in Biology from Rutgers University and is PMP certified.Alexei Saveliev has a 20+ year career in molecular biology with operational roles focused on practical applications of massively parallel sequencing in gene therapy and translational research. Before joining CBM, he was Senior Scientist, NGS Process Development at Azenta Life Sciences where he worked on the implementation of third-generation, long-read sequencing approaches for deep analysis of gene therapy vectors. He was also Associate Director of the newly created Nucleic Acid Technologies Core at the Gene Therapy Program, University of Pennsylvania where he developed comprehensive analytical infrastructure, implemented a novel bioinformatics pipeline for upstream vector production, managed a team of NGS scientists, and contributed to hundreds of individual NGS projects as well as several research papers. Alexei has a Masters in Genetics and a Certificate in Business Administration from The Wharton School.

Gene delivery & gene editing platform evolution

Dr Rodino-Klapac is a gene therapy pioneer who has dedicated her professional life to advancing medicines designed to treat genetically based diseases. With professional experience across industry and academia, she is renowned for her contributions to molecular genetics and gene therapy that have advanced the field. She is author to a vast body of published, peer-reviewed work, the recipient of multiple awards, a National Institutes of Health (NIH) Fellow appointee, and current Board member of the Association for Regenerative Medicine, as well as a member of the American Society for Gene and Cell Therapy, the American Academy of Neurology, and the American Association for the Advancement of Science. She is the former head of the Laboratory for Gene Therapy Research at Nationwide Children’s Hospital, established the Gene Therapy Center of Excellence within Sarepta and leads the Company’s Gene Editing Innovation Center, directing a team of researchers to discover and develop novel gene replacement and gene editing therapies. She co-founded and served as chief scientific officer of Myonexus Therapeutics, a gene therapy company focused on limb-girdle muscular dystrophies acquired by Sarepta in 2019. She currently serves as Sarepta’s Executive Vice President, Chief Scientific Officer. Her work has led to 11 investigational new drug applications and she is the co-inventor of SRP-9001, an investigational micro-dystrophin gene therapy, and the inventor of five investigational gene therapies for limb-girdle muscular dystrophy. She is the inventor of over 50 US and over 70 international published patent applications. She earned her PhD in molecular genetics form the Ohio State University and graduated summa cum laude from Kings College in Wilkes-Barre, Pennsylvania, with a Bachelor of Science degree in Biology.

Antisense transcription from lentiviral gene targeting linked to an integrated stress response in colorectal cancer cells

Advances in gene therapy research have resulted in the successful development of new therapies for clinical use. Here, we explored a gene targeting approach to deplete ephrinB2 from colorectal cancer cells using an inducible lentiviral vector. EphrinB2, a transmembrane ephrin ligand, promotes colorectal cancer cell growth and viability and predicts poor patient survival when expressed at high levels in colorectal cancer tissues. We discovered that lentiviral vector integration and expression in the host DNA frequently drive divergent host gene transcription, generating antisense reads coupled with splicing events and generation of chimeric vector/host transcripts. Antisense transcription of host DNA was linked to development of an integrated stress response and cell death. Despite recent successes, off-target effects remain a concern in genetic medicine. Our results provide evidence that divergent gene transcription is a previously unrecognized off-target effect of lentiviral vector integration with built-in properties for regulation of gene expression.

Gene Editing for Inherited Red Blood Cell Diseases.

Today gene therapy is a real therapeutic option to address inherited hematological diseases that could be beneficial for thousands of patients worldwide. Currently, gene therapy is used to treat different monogenic hematological pathologies, including several red blood cell diseases such as β-thalassemia, sickle cell disease and pyruvate kinase deficiency. This approach is based on addition gene therapy, which consists of the correction of hematopoietic stem cells (HSCs) using lentiviral vectors, which integrate a corrected version of the altered gene. Lentivirally-corrected HSCs generate healthy cells that compensate for the deficiency caused by genetic mutations. Despite its successful results, this approach lacks both control of the integration of the transgene into the genome and endogenous regulation of the therapeutic gene, both of which are important aspects that might be a cause for concern. To overcome these limitations, gene editing is able to correct the altered gene through more precise and safer approaches. Cheap and easy-to-design gene editing tools, such as the CRISPR/Cas9 system, allow the specific correction of the altered gene without affecting the rest of the genome. Inherited erythroid diseases, such as thalassemia, sickle cell disease and Pyruvate Kinase Deficiency, have been the test bed for these gene editing strategies, and promising results are currently being seen. CRISPR/Cas9 system has been successfully used to manipulate globin regulation to re-activate fetal globin chains in adult red blood cells and to compensate for hemoglobin defects. Knock-in at the mutated locus to express the therapeutic gene under the endogenous gene regulatory region has also been accomplished successfully. Thanks to the lessons learned from previous lentiviral gene therapy research and trials, gene editing for red blood cell diseases is rapidly moving from its proof-of-concept to its first exciting results in the clinic. Indeed, patients suffering from β-thalassemia and sickle cell disease have already been successfully treated with gene editing, which will hopefully inspire the use of gene editing to cure erythroid disorders and many other inherited diseases in the near future.

Non-Viral Vectors for Delivery of Nucleic Acid Therapies for Cancer.

The research and development of non-viral gene therapy has been extensive over the past decade and has received a big push thanks to the recent successful approval of non-viral nucleic acid therapy products. Despite these developments, nucleic acid therapy applications in cancer have been limited. One of the main causes of this has been the imbalance in development of delivery vectors as compared with sophisticated nucleic acid payloads, such as siRNA, mRNA, etc. This paper reviews non-viral vectors that can be used to deliver nucleic acids for cancer treatment. It discusses various types of vectors and highlights their current applications. Additionally, it discusses a perspective on the current regulatory landscape to facilitate the commercial translation of gene therapy.

\u201cCRISPR for Disabilities: How to Self-Regulate\u201d or Something?

The development of the CRISPR gene editing technique has been hyped as a technique that could fundamentally change scientific research and its clinical application. Unrecognized is the fact that it joins other technologies that have tried and failed under the same discourse of scientific hype. These technologies, like gene therapy and stem cell research, have moved quickly passed basic research into clinical application with dire consequences. Before hastily moving to clinical applications, it is necessary to consider basic research and determine how CRISPR/Cas systems should be applied. In the case of single gene diseases, that application is expected to have positive impacts, but as we shift to more complex diseases, the impact could be unintentionally negative. In the context of common disabilities, the level of genetic complexity may render this technology useless but potentially toxic, aggravating a social discourse that devalues those with disabilities. This paper intends to define the issues related to disability that are associated with using the CRIPSR/Cas system in basic research. It also aims to provide a decision tree to help determine whether the technology should be utilized or if alternative approaches beyond scientific research could lead to a better use of limited funding resources.

Precision Orthodontics: How Much Can Genomics and Gene Therapy Help?

With the newly emerging concepts and advancements in genetic/genomic technologies, information on causative or candidate gene(s), transcription factors or growth factors responsible for orthodontic cases such as craniofacial dysmorphologies, morphogenesis and differentiation of craniofacial tissues, midfacial defects, crowding of teeth, prognathism, genetic variations associated with susceptibility of developing and/or heterogeneity in treatment response of malocclusions are emerging. Promising results have been accrued on utilization of genetic testing for management of monogenic traits such as primary failure of eruption (PFE), and Class III malocclusion. As the impact of genetic/genomic factors on orthodontic treatment outcome is emerging, gene therapy as a novel approach is being explored for effective and precise orthodontic treatment. Despite certain challenges and biosafety issues, the emergence of some promising success stories and ongoing research in gene therapy provides an optimistic future in the field of precision orthodontics. However, in order for precision orthodontics to be implemented in daily practice, more exquisite research is warranted. This review encompasses a conspectus on the potentials of genomics and gene therapy in achieving precision orthodontics.

CRISPR/Cas-based diagnostic platforms

Over the last few years, CRISPR/Cas systems have been extensively studied and used for a wide range of applied purposes. The variety of their applications is accounted for by the ability of Cas-type nucleases to targetly cleave specified nucleic acid sequences. In this case, the researcher might set the necessary sequence of the guiding elements in the CRISPR/Cas system, played by so-called single guide RNAs allowing it to act on select targets. This potential underlies one of the reasons for exerting interest in CRISPR/Cas systems. One of the first areas for applying these systems was its use for genomic editing. Later, the list of potential opportunities has been expanded: e.g., they can be used in gene therapy and epigenetic research. It is possible to create sgRNA libraries which might be used to create a pool of viral vectors applied for bacterial cell transformation with subsequent cas-protein transduction that cause target gene knockout. This approach allows finding genes responsible for resistance or sensibility to diverse substances. Using such systems in molecular diagnostics of infectious diseases is considered as one of the most promising directions allowing to detect even extremely low concentrations of pathogenic organisms in samples due to their specific nucleotide sequences. Simultaneously, such assays turn out to be accurate, rapid and easy to utilize. In addition, some platforms may work without using expensive equipment, because methods for fast and simple sample preparation have already been developed, whereas modern preamplification approaches allow to avoid applying thermocycling devices. Interestingly, a great amount of diverse types of natural CRISPR/Cas systems have been already discovered. Such abundance promotes development of multiple artificial systems, each of which exerting own unique characteristics. Therefore, a variety of diagnostic platforms with different properties are created on their basis that allows researchers and physicians to choose an optimal approach for performing specific tasks. For this reason, insights into structure and operation of CRISPR/Cas systems are necessary for selecting a suitable platform. The current classification of systems is based on such principles serving as the basis, in turn, for convenient evaluation of the very variety of molecular diagnostics platforms and presentation of the typical technical characteristics and nuances for each method. Thus, this review, which is mainly devoted to the platforms for molecular diagnostics of infectious diseases, also touches upon the issues of functioning, devices, and classification of CRISPR/Cas systems.

Recommendations on ELISpot assay validation by the GCC.

Gene therapy, cell therapy and vaccine research have led to an increased need to perform cellular immunity testing in a regulated environment to ensure the safety and efficacy of these treatments. The most common method for the measurement of cellular immunity has been Enzyme-Linked Immunospot assays. However, there is a lack of regulatory guidance available discussing the recommendations for developing and validating these types of assays. Hence, the Global CRO Council has issued this white paper to provide a consensus on the different validation parameters required to support Enzyme-Linked Immunospot assays and a harmonized and consistent approach to Enzyme-Linked Immunospot validation among contract research organizations.

Improved and Flexible HDR Editing by Targeting Introns in iPSCs

Highly efficient gene knockout (KO) editing of CRISPR–Cas9 has been achieved in iPSCs, whereas homology-directed repair (HDR)-mediated precise gene knock-in (KI) and high-level expression are still bottlenecks for the clinical applications of iPSCs. Here, we developed a novel editing strategy that targets introns. By targeting the intron before the stop codon, this approach tolerates reading frameshift mutations caused by nonhomologous end-joining (NHEJ)-mediated indels, thereby maintaining gene integrity without damaging the non-HDR-edited allele. Furthermore, to increase the flexibility and screen for the best intron-targeting sgRNA, we designed an HDR donor with an artificial intron in place of the endogenous intron. The presence of artificial introns, particularly an intron that carries an enhancer element, significantly increased the reporter expression levels in iPSCs compared to the intron-deleted control. In addition, a combination of the small molecules M3814 and trichostatin A (TSA) significantly improves HDR efficiency by inhibiting NHEJ. These results should find applications in gene therapy and basic research, such as creating reporter cell lines.

Droplette: A Platform Technology to Directly Deliver Nucleic Acid Therapeutics and Other Molecules into Cells and Deep into Tissue Without Transfection Reagents.

Droplette (US 9,700,686 B2 and PCT/US2016/035695) is the first portable and contact-free transdermal technology comprising the unique combination of a piezoelectric transducer and a pneumatic diaphragm pump to deliver large biomolecules including nucleic acid therapeutics (NATs) deep into cells, and into skin and soft tissue for effective delivery over short timescales. The droplets that come out of the device are 10-50× smaller upon impact than what is created through other commercial atomizers, such as the piezoelectric transducer alone. This device has been tested extensively in vitro, in vivo and in IRB approved human studies. The Droplette device delivers metered doses using a water droplet dispersal technology already commonly used in humidifier devices, by utilizing a piezoelectric material. Three key innovations make this device technically novel and tailored specifically for both field and lab use: (1) The combination of the piezo and pump to generate sub-micron drug-loaded droplets that penetrate cells, skin, and soft tissue to effectively deliver a range of large molecules, proteins, and nucleic acids. (2) Their assembly in a modular manner which enables portability, safe sterilization, and ejection without direct device-surface contact or significant force, allowing for improved safety and ease of use in both research and clinical settings. (3) The integration of a single-use, sterile cartridge that contains a therapeutic formulation and allows easy integration of a large number of molecules. The platform has broad applications across multiple fields, such as delivery of drugs for inflammatory skin diseases, antibiotics for skin infections, and gene delivery for gene therapy and biomedical research. In this chapter, we briefly introduce the Droplette delivery technology, instructions for building your own setup (note that fully built devices are available for purchase from Droplette, Inc), and provide protocols for directly transfecting adherent cell cultures and for direct in vivo transfection using the Droplette system without the need for traditional transfection reagents or methods.

Updates on Cardiac Gene Therapy Research and Methods: Overview of Cardiac Gene Therapy.

Gene therapy has made a significant progress in clinical translation over the past few years with several gene therapy products currently approved or anticipating approval for clinical use. Cardiac gene therapy lags behind that of other areas of diseases, with no application of cardiac gene therapy yet approved for clinical use. However, several clinical trials for gene therapy targeting the heart are underway, and innovative research studies are being conducted to close the gap. The second edition of Cardiac Gene Therapy in Methods in Molecular Biology provides protocols for cutting-edge methodologies used in these studies. In this chapter, we discuss recent updates on cardiac gene therapy studies and provide an overview of the chapters in the book.

Egr1/HSP70 Promoter-Driven Activation of Gene Expression for Synergistic Anti-Hepatoma Using PEI-MZF Nanoparticles and Radiation.

IntroductionSpatially restricted gene expression circumvents the gene expression and gene vector problem by enabling localized amplification. The objective of this study is to construct a spatially restricted gene expression for liver cancer therapy based upon the MFH-absorbing properties of PEI- Mn0.5zn0.5Fe2o4, gene therapy and radiation.MethodsMn0.5zn0.5Fe2O4 (MZF) magnetic nanoparticles were prepared by an improved chemical co-precipitation method, modified by polyethylene imine (PEI), and then the structure, modification characters, biocompatibility, temperature rise and control ability and binding efficiency of the plasmid were characterized. Then, the dual-promoter plasmid PCDNA3.1-EGR1-HSP70-HSVTK was constructed. The recombinant vectors were identified by enzyme digestion analysis and DNA sequencing. The TK gene expression level was detected by realtime-PCR assay in HEK293 cells. Also, the HSV-TK gene expression was detected in SMMC7721 cells with the help of PEI-Mn0.5Zn0.5Fe2O4. In vitro anti-tumor experiment, MTT assay and flow cytometry were used to evaluate the therapeutic effects of the cultured SMMC7721 cells treated by different ways. In vivo anti-tumor experiment, the xenografted mice were treated by different ways for three times to detect the antitumor effect.ResultsThe Mn0.5Zn0.5Fe2O4 magnetic nanoparticles could be successfully prepared through improved co-precipitation process and showed good biocompatibility. And PEI had been coated on MZF complex. The modified PEI-MZF presented favorable dispensability, responsibility to magnetism, good loading capability and transfect capability. Also, pCDNA3.1-Egr1-Hsp70-HSVTK plasmid had been constructed successfully and could be induced by heat and irradiation. It would be used for further target gene therapy research. The antitumor results in vitro showed: The therapeutic effects of nanosized PEI-MZF-HSV-TK complex could significantly inhibit the proliferation of cultured liver cancer cells (SMC7721), induce cell apoptosis and had a prominent cell cycle disturbance in the S phase in vitro. The results in vivo showed: The combined therapy induced by PEI-MZF-HSV-TK could inhabit the growth of hepatocellular carcinoma xenografts by killing and inhabiting the proliferation of the tumor cells.ConclusionThe novel site-directed heat/radiation-inducible expression system based upon the hyperthermia (by MFH) and radiation possessed superior antitumor effect in vivo and in vitro.

Targeted Nanotherapeutics Using LACTB Gene Therapy Against Melanoma.

Introductionβ-lactamase (LACTB) is a tumor suppressor gene in various tumors including melanoma. However, it remains challenging to efficiently deliver the LACTB gene into melanoma. Recently, we designed a nonviral nanocarrier iRGD/DOTAP/MPEG-PDLLA (iDPP) that could deliver gene targetedly to melanoma efficiently without obvious adverse effects.MethodsIn this study, the tumor-targeted nanoparticle iDPP was prepared to deliver LACTB gene to treat melanoma in vitro and in vivo. First, the expression level of LACTB in 6 clinical specimens of melanoma patients was evaluated. Subsequently, the characteristics of iDPP/LACTB nanocomplexes were studied. Afterwards, the in vitro and in vivo anti-tumor efficacy of the iDPP/LACTB nanocomplexes were explored utilizing the B16-F10 mouse melanoma cell line and the B16-F10 subcutaneous melanoma model.ResultsCompared with the normal epithelium, the expression level of LACTB in melanoma tissues was significantly downregulated. In vitro B16-F10 cell tests showed iDPP/LACTB nanocomplexes could increase the mRNA levels of P21, Bid, Bax, Pidd1, and Sival genes and up-regulate the p53 signaling pathway of melanoma cells, thus promoting cell apoptosis and blocking the cell cycle. Injected intravenously, iDPP nanoparticles could deliver DNA to the subcutaneous melanoma targetedly. Based on in vivo mouse xenograft model, iDPP/LACTB nanocomplexes could effectively inhibit tumor proliferation and induce tumor apoptosis, thus significantly inhibiting melanoma growth (tumor inhibition rate is about 68%) in the subcutaneous B16-F10 melanoma model.ConclusionThe downregulated LACTB might be a potential target for melanoma therapy. The iDPP/LACTB nanocomplexes could inhibit the growth of the mouse melanoma without obvious side effects, which provide a new option for melanoma gene therapy research.

From insulin injections to islet transplantation: An overview of the journey.

When, in 1869, Paul Langerhans detected the "islands of tissue" in the pancreas, he took the first step on a journey towards islet transplantation as a treatment for type 1 diabetes. The route has embraced developments across biosciences, surgery, gene therapy and clinical research. This review highlights major milestones along that journey involving whole pancreas transplantation, islet transplantation, the creation of surrogate insulin-secreting cells and novel islet-like structures using genetic and bio-engineering technologies. To obviate the paucity of human tissue, pluripotent stem cells and non-β-cells within the pancreas have been modified to create physiologically responsive insulin-secreting cells. Before implantation, these can be co-cultured with endothelial cells to promote vascularisation and with immune defence cells such as placental amnion cells to reduce immune rejection. Scaffolds to contain grafts and facilitate surgical placement provide further opportunities to achieve physiological insulin delivery. Alternatively, xenotransplants such as porcine islets might be reconsidered as opportunities exist to circumvent safety concerns and immune rejection. Thus, despite a long and arduous journey, the prospects for increased use of tissue transplantation to provide physiological insulin replacement are drawing ever closer.

Longitudinal clonal tracking in humanized mice reveals sustained polyclonal repopulation of gene-modified human-HSPC despite vector integration bias

BackgroundCurrent understanding of hematopoiesis is largely derived from mouse models that are physiologically distant from humans. Humanized mice provide the most physiologically relevant small animal model to study human diseases, most notably preclinical gene therapy studies. However, the clonal repopulation dynamics of human hematopoietic stem and progenitor cells (HSPC) in these animal models is only partially understood. Using a new clonal tracking methodology designed for small sample volumes, we aim to reveal the underlying clonal dynamics of human cell repopulation in a mouse environment.MethodsHumanized bone marrow-liver-thymus (hu-BLT) mice were generated by transplanting lentiviral vector-transduced human fetal liver HSPC (FL-HSPC) in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice implanted with a piece of human fetal thymus. We developed a methodology to track vector integration sites (VIS) in a mere 25 µl of mouse blood for longitudinal and quantitative clonal analysis of human HSPC repopulation in mouse environment. We explored transcriptional and epigenetic features of human HSPC for possible VIS bias.ResultsA total of 897 HSPC clones were longitudinally tracked in hu-BLT mice—providing a first-ever demonstration of clonal dynamics and coordinated expansion of therapeutic and control vector-modified human cell populations simultaneously repopulating in the same humanized mice. The polyclonal repopulation stabilized at 19 weeks post-transplant and the contribution of the largest clone doubled within 4 weeks. Moreover, 550 (~ 60%) clones persisted over 6 weeks and were highly shared between different organs. The normal clonal profiles confirmed the safety of our gene therapy vectors. Multi-omics analysis of human FL-HSPC revealed that 54% of vector integrations in repopulating clones occurred within ± 1 kb of H3K36me3-enriched regions.ConclusionsHuman repopulation in mice is polyclonal and stabilizes more rapidly than that previously observed in humans. VIS preference for H3K36me3 has no apparent negative effects on HSPC repopulation. Our study provides a methodology to longitudinally track clonal repopulation in small animal models extensively used for stem cell and gene therapy research and with lentiviral vectors designed for clinical applications. Results of this study provide a framework for understanding the clonal behavior of human HPSC repopulating in a mouse environment, critical for translating results from humanized mice models to the human settings.

A Changing World in Gene Therapy Research: Exciting Opportunities for Medical Advancement and Biosafety Challenges.

Introduction: We previously reported on the United States' regulatory environment evolving to accommodate an emerging boom in gene therapy research. Several important developments have transpired in the 2 years since that article was published, including the coronavirus disease 2019 (COVID-19) pandemic and the drive for large-scale testing of vaccines containing recombinant or synthetic nucleic acid molecules. This report highlights key developments in the field with a focus on biosafety and issues of note to biosafety professionals with responsibilities over clinical research.Discussion: We provide guidance for performing risk assessments on the currently approved gene therapy products as well as the most utilized types of investigational products in clinical trials. Areas of focus include the prominent approaches utilized in the three major areas of research: oncology, infectious diseases, and rare diseases.Conclusion: The COVID-19 pandemic has created several opportunities for continued growth in gene therapy. National vaccination campaigns will result in greater public acceptance of gene therapy research. Technological advancements that made the vaccine race possible will spur the next generation of research. Advancements born in the developed world set the stage for the creation of therapeutics to treat greater numbers in the developing world and have the potential for massive benefits to global public health. Biosafety professionals and Institutional Biosafety Committees play key roles in contributing to the safe evidence-based advancement of gene therapy research. Biosafety professionals responsible for clinical research oversight must be aware of emerging technologies and their associated risks to support the safe and ethical conduct of research.