The objective of this article is to summarize the research progress and discuss the current difficulties of gene-based therapeutic angiogenesis in lower limb ischemic diseases, so as to provide new research directions fo...The objective of this article is to summarize the research progress and discuss the current difficulties of gene-based therapeutic angiogenesis in lower limb ischemic diseases, so as to provide new research directions for the non-invasive treatment of lower limb ischemia. The basic and clinical trials of gene-based therapeutic angiogenesis in lower limb ischemia in recent years were read and reviewed. Growth factors such as vascular endothelial growth factor, hepatocyte growth factor, and fibroblast growth factor have been extensively studied for their application in lower limb ischemic diseases. However, clinical studies across various phases have shown inconsistent efficacy endpoints. The efficacy of gene therapy remains questionable. Before exploring efficient methods of delivering pro-angiogenic genes to ischemic tissues, clarification is needed regarding whether the goal of gene therapy is to simply promote collateral circulation or create a conducive tissue microenvironment for angiogenesis. In conclusion, pre-clinical and clinical studies have demonstrated the potential of therapeutic angiogenesis, but more systematic and comprehensive research is needed to explore safer, more effective, and cost-effective treatment methods.
The advent of genome editing has kindled the hope to cure previously uncurable, life-threatening genetic diseases. However, whether this promise can be ultimately fulfilled depends on how efficiently gene editing agents...The advent of genome editing has kindled the hope to cure previously uncurable, life-threatening genetic diseases. However, whether this promise can be ultimately fulfilled depends on how efficiently gene editing agents can be delivered to therapeutically relevant cells. Over time, viruses have evolved into sophisticated, versatile, and biocompatible nanomachines that can be engineered to shuttle payloads to specific cell types. Despite the advances in safety and selectivity, the long-term expression of gene editing agents sustained by viral vectors remains a cause for concern. Cell-derived vesicles (CDVs) are gaining traction as elegant alternatives. CDVs encompass extracellular vesicles (EVs), a diverse set of intrinsically biocompatible and low-immunogenic membranous nanoparticles, and virus-like particles (VLPs), bioparticles with virus-like scaffold and envelope structures, but devoid of genetic material. Both EVs and VLPs can efficiently deliver ribonucleoprotein cargo to the target cell cytoplasm, ensuring that the editing machinery is only transiently active in the cell and thereby increasing its safety. In this review, we explore the natural diversity of CDVs and their potential as delivery vectors for the clustered regularly interspaced short palindromic repeats (CRISPR) machinery. We illustrate different strategies for the optimization of CDV cargo loading and retargeting, highlighting the versatility and tunability of these vehicles. Nonetheless, the lack of robust and standardized protocols for CDV production, purification, and quality assessment still hinders their widespread adoption to further CRISPR-based therapies as advanced "living drugs." We believe that a collective, multifaceted effort is urgently needed to address these critical issues and unlock the full potential of genome-editing technologies to yield safe, easy-to-manufacture, and pharmacologically well-defined therapies. Finally, we discuss the current clinical landscape of lung-directed gene therapies for cystic fibrosis and explore how CDVs could drive significant breakthroughs in gene editing for this disease.
Herzog CR, Zhang J, Feng X
… +12 more, Dang TT, Yu X, Huang J, Fang F, Gao H, Yu X, Wang Y, Han R, Liu Y, Cornetta K, Xiao W, Xu W
Hum Gene Ther
· 2025 May · PMID 40293710
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Human embryonic kidney 293 (HEK 293) cells are the main producer cell line for recombinant adeno-associated virus (rAAV) production. However, AAV vector yields among 293 clones vary considerably. To elucidate the biologi...Human embryonic kidney 293 (HEK 293) cells are the main producer cell line for recombinant adeno-associated virus (rAAV) production. However, AAV vector yields among 293 clones vary considerably. To elucidate the biological basis for these differences, whole genomes of an adherent and a suspension 293 cell clone with high-yield rAAV were sequenced using nanopore technology. All 293 cell derivative lines showed a twofold copy number gain at the adenoviral integration site across, suggesting a genome duplication event. To our surprise, the two high-producer clones, despite having been separately developed, are biologically closely grouped together as compared to other commonly used 293 clones. Their genomes contain a similar adenoviral gene integration region, which likely leads to high expression of proteins that facilitate AAV replication and packaging. Thus, genome duplication in the adenovirus integration locus may be a key factor affecting AAV production yield.
Over the last two decades, adeno-associated viruses (AAVs) have been widely used as viral vectors in gene therapy due to their ability to infect both dividing and nondividing cells, broad tissue specificity, and favorabl...Over the last two decades, adeno-associated viruses (AAVs) have been widely used as viral vectors in gene therapy due to their ability to infect both dividing and nondividing cells, broad tissue specificity, and favorable safety profile. Recombinant AAV (rAAV) production requires a helper virus, typically adenovirus (AdV), which provides essential genes for AAV replication. However, the increasing demand for safer and more efficient rAAV production methods led to the need to develop helper plasmids with minimal AdV components. In this study, we evaluate the impact of AdV E2 and E4 in the productivity and genome packaging of rAAV serotypes 2, 5, 8, and 9, produced by transient transfection. We designed and tested eight novel helper plasmids with different deletions in E2 and E4 genes. Results indicated that deletions in these genes significantly affected rAAV productivity and packaging, particularly for serotypes 8 and 9. Helper plasmids containing minimal essential genes-E2-DBP, E4orf6, and VA RNA-showed near to 10-fold reduction in viral genome packaging compared to the control. However, including E2 L4-22/33K and E4orf3 regions significantly improved viral production, particularly for serotypes 8, and 9. In this study, we also demonstrated that the full E4 gene is crucial to achieving high full-empty ratios, minimizing the production of empty capsids, and enhancing viral release into the culture medium of rAAV8. Accordingly, we created a smaller plasmid, without adenoviral structural proteins that allows a similar rAAV production across all tested serotypes. Overall, these findings provide insights into the genetic requirements for efficient rAAV production and highlight the importance of the E2 and E4 regions for optimizing viral yield and quality. This approach could lead to the development of improved strategies for large-scale rAAV vector production by enabling safer and more cost-effective systems.
The development of potency assays for Advanced Therapy Medicinal Products (ATMPs) presents significant challenges due to the variability of starting materials and the complex mechanisms of action involved. This article a...The development of potency assays for Advanced Therapy Medicinal Products (ATMPs) presents significant challenges due to the variability of starting materials and the complex mechanisms of action involved. This article aims to address the following key question: To answer this, we employed a mixed-methods approach, synthesizing data from scientific literature, industry reports, and regulatory guidelines to identify current limitations and innovative solutions for potency assay development. Our methodology integrates a systematic review of academic publications (2018-2024) to capture recent advancements in biotechnology and their applicability to potency testing. We complemented this with an analysis of industry perspectives, drawn from webinars and white papers, as well as a detailed comparison of global regulatory frameworks, including the FDA's new guidance on potency assurance for Cellular and Gene Therapy Products (CGTs/ATMPs). Additionally, we developed a comprehensive database to analyze potency assays used in approved, rejected, and withdrawn CGT/ATMP products, focusing on technical and regulatory challenges. Based on this multilevel analysis, we propose a product-specific framework for designing, developing, and validating potency assays for different ATMP categories, taking into account their unique technical and regulatory constraints. We also highlight emerging technologies, such as droplet digital polymerase chain reaction and reporter gene assays, as innovative tools for improving the precision and reliability of potency testing. Our findings underscore the need for flexible, risk-based strategies in potency assay development that evolve throughout product development and clinical trial phases. Future recommendations emphasize assay standardization, the definition of acceptable variability, and stronger correlations between in vitro potency data and clinical outcomes.
Weiß M, Selig M, Friedrich J
… +12 more, Wierczeiko A, Diederich S, Sigel H, Bredow J, Eichler FS, Nagy A, Seyler D, Holthöfer L, Gerber S, Schweiger S, Linke M, Bley A
Hum Gene Ther
· 2025 Sep · PMID 40257001
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Canavan disease (CD) is a rare autosomal recessive leukodystrophy caused by biallelic pathogenic variants in the gene. CD is characterized by developmental delay, macrocephaly, and abnormal muscle tone. The biochemical...Canavan disease (CD) is a rare autosomal recessive leukodystrophy caused by biallelic pathogenic variants in the gene. CD is characterized by developmental delay, macrocephaly, and abnormal muscle tone. The biochemical diagnosis is confirmed by increased -acetylaspartic acid levels. The phenotypic presentation varies, with 85-90% of individuals exhibiting the severe, typical form, while 10-15% present with a milder, atypical form. Here we report on five patients with a clinical and biochemically proven diagnosis in whom a second pathogenic variant had not yet been identified. Targeted long-read sequencing of the entire gene revealed an SVA_E retrotransposable element located in intron 4 that had been missed by standard short-read-based diagnostic procedures. Haplotype analysis of all patients showed linkage of the SVA_E element with a noncoding variant in intron 1. Functional characterization of the SVA_E element suggests that transcripts of the affected allele are prone to highly efficient mRNA degradation processes. These findings enhance the precision of genetic diagnostics and enable improved guidance for families as well as facilitating potential access to targeted therapies.
Adeno-associated virus (AAV)-based therapeutics have the potential to transform the lives of patients by delivering one-time treatments for a variety of diseases. However, a critical challenge to their widespread adoptio...Adeno-associated virus (AAV)-based therapeutics have the potential to transform the lives of patients by delivering one-time treatments for a variety of diseases. However, a critical challenge to their widespread adoption and distribution is the high cost of goods. Reducing manufacturing costs by developing AAV capsids with improved yield, or fitness, is key to making gene therapies more affordable. AAV fitness is largely determined by the amino acid sequence of the capsid, however, engineered AAVs are rarely optimized for manufacturability. Here, we report a state-of-the art machine learning (ML) model that predicts the fitness of AAV2 capsid mutants based on the amino acid sequence of the capsid monomer. By combining a protein language model (PLM) and classical ML techniques, our model achieved a significantly high prediction accuracy (Pearson correlation = 0.818) for capsid fitness. Importantly, tests on completely independent datasets showed robustness and generalizability of our model, even for multimutant AAV capsids. Our accurate ML-based model can be used as a surrogate for laborious experiments, thus saving time and resources, and can be deployed to increase the fitness of clinical AAV capsids to make gene therapies economically viable for patients.
The underlying risk of retroviral vector-induced insertional oncogenesis in gene therapies requires a reliable preclinical safety assessment. Dysregulation of genes neighboring the vector's integration sites has triggere...The underlying risk of retroviral vector-induced insertional oncogenesis in gene therapies requires a reliable preclinical safety assessment. Dysregulation of genes neighboring the vector's integration sites has triggered hematopoietic malignancies in patients treated with different vector genera and designs. With ca. 18 years in practical use, the immortalization (IVIM) assay can quantify this mutagenic potential and is actively requested by regulatory authorities during preclinical stages. Here, we present a thorough meta-analysis of IVIM data alongside a step-by-step cell culture protocol. On this basis, we propose clonal outgrowth as the single indicator of mutagenicity, simplifying the IVIM assay cost- and time-wise.
β-thalassemia and sickle cell disease (SCD) are global monogenic blood system disorders, and reactivated δ-globin is expected to replace missing or abnormal β-globin. With the development of gene editing technology, acti...β-thalassemia and sickle cell disease (SCD) are global monogenic blood system disorders, and reactivated δ-globin is expected to replace missing or abnormal β-globin. With the development of gene editing technology, activating γ-globin for treating β-thalassemia and SCD has been highly successful. However, δ-globin, as another important potential therapeutic target, has few related studies. Gene editing technology introduced cis-acting elements, including NF-Y, KLF1, GATA1, and TAL1, into the regulatory region of , successfully activating the expression of δ-globin. It was confirmed that the activation effect of δ-globin was closely related to the location of the introduced cis-acting elements. In this study, the mutation creates a de novo binding site for KLF1 at -85∼93 bp upstream of the transcription start site of the gene, as well as the site for TAL1 and GATA1 cobinding motifs at -59 to ∼-78 bp, which could effectively activate δ-globin.
Wacker BK, Bi L, Saenz-Pipaon G
… +6 more, Sanford N, Regan AZ, Lim NS, Liu L, Kim F, Dichek DA
Hum Gene Ther
· 2025 Apr · PMID 40111153
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Endothelial activation and dysfunction are key early steps in atherogenesis. Vascular gene therapy targeting endothelial inflammation and cholesterol accumulation could decrease atherosclerosis progression. ATP-binding c...Endothelial activation and dysfunction are key early steps in atherogenesis. Vascular gene therapy targeting endothelial inflammation and cholesterol accumulation could decrease atherosclerosis progression. ATP-binding cassette subfamily A member 1 (ABCA1) exhibits anti-inflammatory properties and promotes cholesterol efflux. A mouse model showed that systemic endothelial overexpression of ABCA1 decreased diet-induced atherosclerosis. To test if local ABCA1 endothelial overexpression protects against atherosclerosis, we used helper-dependent adenoviral vectors (HDAd) to express ABCA1 or a "Null" control in the carotid endothelium of hyperlipidemic rabbits. Both mRNA and endothelial protein were increased 3 days after vector infusion. After 24 weeks on a high-fat diet, laser-microdissected endothelium showed increased mRNA expression, but whole-vessel mRNA was decreased with HDAdABCA1. Endothelial ABCA1 protein could not be measured at 24 weeks, so its overexpression may be transient. expression was decreased (-23%, < 0.001), but (-15%, = 0.3) was unchanged. Macrophage markers for both M1-like macrophages (: -44% [ = 0.02]; : -40% [ = 0.02]; : -25% [ = 0.02]) and M2-like macrophages (: -27% [ = 0.03]; : -23% [ = 0.09]; : -13% [ < 0.001]) were also decreased. The inflammatory cytokines (-100%; < 0.001) and ( < 0.05) were significantly decreased in the laser-microdissected endothelium, but (+5%, = 1.0) was unchanged and (+101%; = 0.03) increased. Lesion size, intimal lipid, and intimal macrophage content were all unchanged ( > 0.5 for all), and vascular cholesterol measured by mass spectrometry (-11%; = 0.9) also showed no difference. There was a small decrease in the intimal/medial ratio. scRNAseq revealed that vector transcripts were not restricted to endothelial cells after 24+ weeks but were detected in most cell types. The exception was modulated smooth muscle cells, which were found in substantial numbers in larger lesions. Overall, transient overexpression of ABCA1 in the vascular endothelium subtly alters the expression of inflammatory markers, providing only a modest atheroprotection.
Recombinant adeno-associated viruses (rAAVs) are widely used viral vectors in human gene therapy. However, DNA impurities, such as plasmid DNA and host cell DNA, remain a significant quality control concern for final pro...Recombinant adeno-associated viruses (rAAVs) are widely used viral vectors in human gene therapy. However, DNA impurities, such as plasmid DNA and host cell DNA, remain a significant quality control concern for final products. Our study examined purified rAAV1-ZsGreen1, rAAV2-ZsGreen1, rAAV5-ZsGreen1, and rAAV6-ZsGreen1 samples and found that they contained 0.69-3.27% DNA impurities derived from three plasmids, as detected by droplet digital PCR. These plasmid-derived impurities primarily consisted of those derived from the pAAV plasmid (≥98.88%), with small amounts of pRC1, pRC2mi342, pRC5, or pRC6 (≤0.91%), and pHelper (≤0.21%) plasmids. To determine the DNA strand form of these impurities within the capsids, we used two different DNases with distinct substrate specificities. The extracted DNA impurities from the rAAV samples exhibited high sensitivity to nuclease P1 but not to lambda exonuclease. Similarly, host cell DNA encapsulated within the capsids revealed similar sensitivities to the nucleases. These findings indicate that DNA impurities derived from the plasmids and host cell DNA are encapsulated into rAAV capsids as single-stranded DNA, likely through a mechanism similar to that of the rAAV genome.
Hum Gene Ther
· 2025 Apr · PMID 40103557
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Gene therapy has emerged as a promising therapeutic avenue, offering targeted treatments for various diseases. Purification of viral vectors presents a pivotal challenge, demanding the removal of impurities while preserv...Gene therapy has emerged as a promising therapeutic avenue, offering targeted treatments for various diseases. Purification of viral vectors presents a pivotal challenge, demanding the removal of impurities while preserving integrity and potency. During manufacturing, producer cells in transfection systems can be transiently transfected or retro-infected by the viral vectors they have just produced-a process referred to as "retro-transduction"-leading them to express the transgenes of interest. This can be a significant source of contamination in the viral solution pool, particularly when the transgenes encode extracellular, secreted proteins, resulting in cytotoxicity and reduced viral potency. Herein, we aimed to evaluate the efficiency of different viral purification systems commonly used in academic and industry settings in removing the transgene background from viral solutions. The efficiency of each system was assessed based on the levels of the secreted transgene (GLuc), which can be quickly detected in a solution and served as a readout for transgene background contamination in the viral pool during downstream processing. Through a systematic evaluation of purification methods, we identified the most effective approaches for producing pure viral batches with minimal transgene background, all while preserving viral potency and functionality. Our study revealed superior performance of batches that underwent purification via tangential flow filtration, which yielded over 90% reduction in GLuc background and the highest transduction efficiency rates. This work provides significant insights for advancing gene therapy applications that rely on the production of viral vectors encoding secreted transgenes.
Mylvara AV, Davidson CD, Alexander D
… +2 more, Venditti CP, Porter FD
Hum Gene Ther
· 2025 Apr · PMID 40014475
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Niemann-Pick disease, type C1 (NPC1), is a rare, fatal neurodegenerative disorder caused by pathological variations in . We and others have previously demonstrated the efficacy of systemic adeno-associated virus (AAV) ge...Niemann-Pick disease, type C1 (NPC1), is a rare, fatal neurodegenerative disorder caused by pathological variations in . We and others have previously demonstrated the efficacy of systemic adeno-associated virus (AAV) gene therapy with AAV9 in murine models of NPC1. The presence of neutralizing antibodies (NAbs) caused by natural exposure to wildtype AAVs may impair AAV transduction efficacy and reduce or negate the benefit of gene therapy. In addition, there remains the question of whether individuals seroconvert with age and whether seroconversion limits the window of therapeutic efficacy. Thus, we assessed the prevalence of anti-AAV9 and anti-AAV2 NAbs in serum samples from 22 individuals with NPC1 at two different time points: one closer to diagnosis (0.9-17 years old) and another collected between 4 and 15 years later during follow-up (6-28 years old). At a titer of <1:5, we found that more than half of the cohort lacked NAbs against either AAV2 (68.2%) or AAV9 (59.1% at time 1, 63.6% at time 2). Notably, only 3 out of 22 individuals showed a transition from undetectable to detectable NAb titers, and most participants maintained stable titers over time, unaffected by age. These data support the feasibility of systemic or direct CNS AAV9 gene therapy in this patient population.
Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder caused by pathogenic variants in the gene encoding iduronate-2-sulfatase (IDS), which hydrolyzes sulfate groups in dermatan sulfate and he...Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder caused by pathogenic variants in the gene encoding iduronate-2-sulfatase (IDS), which hydrolyzes sulfate groups in dermatan sulfate and heparan sulfate. The current treatment for MPS II includes enzyme replacement therapy and hematopoietic stem cell transplantation (HSCT). Both therapies have shown limited penetration through the blood-brain barrier. Anecdotal cases have been reported with the HSCT benefit to treat neurological problems in MPS II. Herein, we generated an MPS II mouse model using CRISPR/Cas9 to examine the effectiveness of CNS-directed, adeno-associated virus (AAV)2/9-mediated human IDS gene transfer in expressing sustained IDS and improving behavior performance in this model. The intracerebroventricular administration of AAV2/9-hIDS showed higher IDS activity in the central nervous system and better auditory function compared with those by intravenous administration. The results provide a strong proof of concept for the clinical translation of our approach to treating patients with MPS II and cognitive impairment.
Hum Gene Ther
· 2025 Oct · PMID 39989340
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The eye is an ideal target for gene therapy due its accessibility, immune privilege, small size, and compartmentalization. Adeno-associated virus (AAV) is the gold standard vector for gene delivery and can be injected vi...The eye is an ideal target for gene therapy due its accessibility, immune privilege, small size, and compartmentalization. Adeno-associated virus (AAV) is the gold standard vector for gene delivery and can be injected via multiple routes of administration to target different parts of this organ. The approval of Luxturna™, a subretinally delivered gene therapy for -associated Leber's congenital amaurosis, and the large number of successful proof of concept studies performed in animal models injected great momentum into the pursuit of additional AAV-based gene therapies for the treatment of retinal disease. This review provides a comprehensive summary of all subretinally, intravitreally, and suprachoroidally delivered AAV-based ocular gene therapies that have progressed to clinical stage. Attention is given to primary (safety) and secondary (efficacy) outcomes, or lack thereof. Lessons learned and future directions are addressed, both of which point to optimism that the ocular gene therapy field is poised for continued momentum and additional regulatory approvals.
Beta-propeller protein-associated neurodegeneration (BPAN) is an ultra-rare, X-linked dominant, neurodevelopmental, and neurodegenerative disease caused by loss-of-function mutations in the gene. It manifests in neurode...Beta-propeller protein-associated neurodegeneration (BPAN) is an ultra-rare, X-linked dominant, neurodevelopmental, and neurodegenerative disease caused by loss-of-function mutations in the gene. It manifests in neurodevelopmental delay and seizures followed by secondary neurological decline with dystonia/parkinsonism and dementia in adolescence and early adulthood and is characterized by progressive accumulation of iron in the basal ganglia. encodes β-propeller-shaped scaffold protein, or WD repeat domain phosphoinositide-interacting protein 4 (WIPI4), which plays an important role in autophagosome formation. While the mechanisms of how WIPI4 loss of function results in neurological decline and brain pathology have not yet been established, findings of lower autophagic activity provide a direct link between impaired autophagy and neurological disease in BPAN. Here we performed phenotypical characterization of a novel mouse model of BPAN, Wdr45_ex9+1g>a mouse. We identified hyperactive behavior and reduction of autophagy markers in brain tissue in Wdr45_ex9+1g>a hemizygous males as early as at 2 months of age. Given the early onset and spectrum of neurological symptoms such as hyper-arousal and attention deficits in human patients, this model presents a disease-relevant phenotype and can be used in preclinical studies. We used this mouse model for a proof-of-concept study to evaluate whether adeno-associated virus (AAV)-mediated central nervous system (CNS)-targeted gene transfer of can provide therapeutic benefit and be considered a therapeutic paradigm for BPAN. We observed successful expression of human transcripts and WIPI4 protein in the brain tissue, rescue of hyperactive behavior, and correction of autophagy markers. These data demonstrate that gene transfer can be a promising therapeutic strategy for BPAN.
Lomash RM, Dehdashti J, Shchelochkov OA
… +15 more, Chandler RJ, Li L, Manoli I, Sloan JL, Terse P, Xu X, Saade D, Stan R, PaVe-GT team, Brooks PJ, Lo DC, Bönnemann CG, Venditti CP, Pariser AR, Ottinger EA
Hum Gene Ther
· 2025 Mar · PMID 39976996
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Gene therapy development presents multiple challenges, and early planning is vital in the successful implementation of such programs. The Platform Vector Gene Therapy (PaVe-GT) program is a National Institutes of Health...Gene therapy development presents multiple challenges, and early planning is vital in the successful implementation of such programs. The Platform Vector Gene Therapy (PaVe-GT) program is a National Institutes of Health (NIH) initiative developing adeno-associated virus (AAV) gene therapies for four low-prevalence rare diseases. Utilizing the platform-based approach, the program aims to incorporate efficiencies throughout the preclinical and clinical development processes followed by public dissemination of scientific and regulatory learnings. Early in development, the establishment of a Target Product Profile (TPP) by the research team is a critical step to guide product development and align preclinical studies to clinical objectives. Based on the specific needs of the investigational product as defined in the TPP, an overall regulatory strategy can then be outlined to meet the regulatory requirements for the first-in-human clinical trials. During the preclinical phase of development, sponsors may request meetings with the Food and Drug Administration (FDA) to gather feedback on the planned studies and regulatory strategy. To pave the way for PaVe-GT's first investigational AAV gene therapy lead candidate, AAV9-hPCCA, we sought early feedback from the FDA utilizing an INitial Targeted Engagement for Regulatory Advice on CBER/CDER ProducTs (INTERACT) meeting. Here, we elaborate on the value of establishing a TPP and the FDA INTERACT meeting by including our initial AAV9-hPCCA TPP, detailing our INTERACT meeting experience, providing all corresponding regulatory documentation, and highlighting lessons learned. The regulatory documents along with templates developed by our program can also be found on the PaVe-GT website (https://pave-gt.ncats.nih.gov/). This communication aims to provide stakeholders with resources that can be applied to drug development programs in establishing a viable regulatory path to clinical trial initiation.
Lentivirus (LV) vectors offer permanent delivery of therapeutic genes to the host through an RNA intermediate genome. They are one of the most commonly used vectors for clinical gene therapy of inherited disorders such a...Lentivirus (LV) vectors offer permanent delivery of therapeutic genes to the host through an RNA intermediate genome. They are one of the most commonly used vectors for clinical gene therapy of inherited disorders such as immune deficiencies and cancer immunotherapy. One of the most difficult challenges facing their widespread application to patients is the large-scale production of highly pure vector stocks. To improve vector production and downstream purification, there has been a recent investment in the United Kingdom to establish good manufacturing process (GMP)-licensed centers for manufacture and quality control. Other requirements for these vectors include their target cell specificity and tropism, how to regulate gene expression of the therapeutic payload and their potential side effects. Comprehensive detail on the full nucleic acid content of LV is unknown, even though they have entered clinical trials. With potential adverse effects in mind, it is important to identify these contents to assess their safety and purity. In this study, we used highly sensitive PacBio long-distance, next-generation sequencing of reverse-transcribed vector component RNA to investigate the nucleic acid composition of recombinant HIV-1 particles generated by human 293T packaging cells. In this article, we describe our findings of nucleic acids other than the recombinant vector genome that exist, which could potentially be delivered during gene transfer, and suggest that removal of these unwanted components be considered before clinical LV application.