Lentiviral Vectors For Gene Therapy Research Articles

A novel high-titer, bifunctional lentiviral vector for autologous hematopoietic stem cell gene therapy of sickle cell disease

A major limitation of gene therapy for sickle cell disease (SCD) is the availability and access to a potentially curative one-time treatment, due to high treatment costs. We have developed a high-titer bifunctional lentiviral vector (LVV) in a vector backbone that has reduced size, high vector yields, and efficient gene transfer to human CD34+ hematopoietic stem and progenitor cells (HSPC). This LVV contains locus control region cores expressing an anti-sickling βAS3-globin gene and two microRNA adapted short hairpin RNA simultaneously targeting BCL11A and ZNF410 transcripts to maximally induce fetal hemoglobin (HbF) expression. This LVV induces high levels of anti-sickling hemoglobins (HbAAS3 + HbF), while concurrently reducing sickle hemoglobin (HbS). The reduction in HbS and increased anti-sickling hemoglobin impedes deoxygenated HbS polymerization and red blood cell sickling at low vector copy per cell in transduced SCD patient CD34+ cells differentiated into erythrocytes. The dual alterations in red cell hemoglobins ameliorated the SCD phenotype in the SCD Berkeley mouse model in vivo. With high titer and enhanced transduction of HSPC at low multiplicity of infection, this LVV will increase the number of patient doses of vector from production lots to reduce costs and help improve accessibility to gene therapy for SCD.

Microglia cells treated with synthetic vasoactive intestinal peptide or transduced with LentiVIP protect neuronal cells against degeneration.

A common pathological hallmark of neurodegenerative disorders is neuronal cell death, accompanied by neuroinflammation and oxidative stress. The vasoactive intestinal peptide (VIP) is a pleiotropic peptide that combines neuroprotective and immunomodulatory actions. The gene therapy field shows long-term promise for treating a wide range of neurodegenerative diseases (ND). In this study, we aimed to investigate the in vitro efficacy of transduction of microglia using lentiviral gene therapy vectors encoding VIP (LentiVIP). Additionally, we tested the protective effects of the secretome derived from LentiVIP-infected "immortalized human" microglia HMC3 cells, and cells treated with Synthetic VIP (SynVIP), against toxin-induced neurodegeneration. First, LentiVIP, which stably expresses VIP, was generated and purified. VIP secretion in microglial conditioned media (MG CM) for LentiVIP-infected HMC3 microglia cells was confirmed. Microglia cells were activated with lipopolysaccharide, and groups were formed as follows: 1) Control, 2) SynVIP-treated, or 3) LentiVIP-transduced. These MG CM were applied on an in vitro neurodegenerative model formed by differentiated (d)-SH-SY5Y cells. Then, cell survival analysis and apoptotic nuclear staining, besides measurement of oxidative/inflammatory parameters in CM of cells were performed. Activated MG CM reduced survival rates of both control and toxin-applied (d)-SH-SY5Y cells, whereas LentiVIP-infected MG CM and SynVIP-treated ones exhibited better survival rates. These findings were supported by apoptotic nuclear evaluations of (d)-SH-SY5Y cells, alongside oxidative/inflammatory parameters in their CM. LentiVIP seems worthy of further studies for the treatment of ND because of the potential of gene therapy to treat diseases effectively with a single injection.

Gene Therapy Expressing Platelet-Derived Factor VIII for Correction of Hemophilia Α with a History of Inhibitors

Introduction: Platelet targeted gene therapy with human factor VIII has established hemostasis in murine and canine models with severe hemophilia A withouteliciting inhibitory antibodies. Here-in we describe feasibility, safety and efficacy of the first subject treated on a first-in-human phase 1 trial that targets factor FVIII synthesis and storage within platelet α-granules for delivery at the site of vascular injury for hemophilia A (NCT03818763). Method: Eligible are severe hemophilia A patients ≥18 years with a history of inhibitors to factor VIII (≥0.6 Bethesda Units [BU]/ml). Following mobilization, autologous CD 34+ cells (≥6 x 10 6/kg) are collected and transduced with a lentiviral vector encoding the ITGA2B gene promoter for ectopic expression of human B-domain-deleted factor VIII within the megakaryocyte lineage. The transduced cells are infused after reduced intensity cytoreduction with fludarabine (120 mg/m 2) and melphalan (120 mg/m 2) followed by a washout period of ≥24 hours. The primary endpoints of feasibility and safety are defined as: 1) feasibility of the cell manufacturing procedure by the availability of ≥4 x 10 6/kg transduced clinical grade CD34+cells, cell viability ≥70% and undetectable microbial contamination and 2) safety defined as hematopoietic recovery ≤28 days of infusion and absence of ≥ grade 3 toxicity (CTCAE version 5.0). Outcome: Subject 1 is a 29-year-old male with severe hemophilia A who developed inhibitors to factor VIII (2.6 BU/ml) in his first year of life. Immune tolerance to factor VIII had been established with a non-detectable inhibitor titer at enrollment. Hemostatic prophylaxis was maintained with emicizumab weekly and recombinant factor VIII for breakthrough bleeding. His annualized bleeding rate was 16 in the 12-month period preceding infusion. The cell product was the result of 1 day of collection yielding 6.73 x 10 6 /kg total viable CD34+ cells post-transduction and vector copy number by qPCR of 1.16 copies/cell. Megakaryocyte factor VIII:C levels were 0.00 mU/10 6 before transduction and 101.32 mU/10 6 after transduction. The cell product satisfied release criteria. Neutrophil recovery (≥0.5 x 10 9/L) and platelet transfusion independence (≥50 x 10 9/L) was achieved 15 days post-infusion and sustained for >12 months. The duration of hospitalization was 21 days with no re-admissions. There was no breakthrough bleeding during collection and hospitalization. During and post-infusion there was no unexpected toxicity ≥ grade 1. He has not required immune suppression. Emicizumab was discontinued 3.6 months post-infusion after demonstration of whole blood vector copy number at 1 and 3-months. There has been no spontaneous bleeding or a need for “on demand” factor VIII after discontinuing emicizumab. Secondary outcomes are summarized in Table 1. No replication competent lentivirus was detected through month 12 post-infusion. Integration site analysis was carried out, and none of the samples tested through 12 months contained cell clones which exceeded 20% relative abundance. Transduced cells were highly polyclonal. Integration site analysis showed no enrichment of integration near cancer-associated genes in the cell product nor in whole blood cell lineages post-infusion. Conclusion: We report feasibility, safety and efficacy in the first subject with severe hemophilia A and a history of inhibitors who received lentiviral vector gene therapy directed to induce megakaryocytes to synthesize and store factor VIII within platelets. These findings extend the potential to treat severe hemophilia A patients who are not eligible for valoctcogene roxaoarvovec (AAV5-hFVIII-SQ).

Exploring nutrient supplementation and bioprocess optimization to improve the production of lentiviral vectors in serum-free medium suspension cultures.

The use of lentiviral vectors (LV) in gene therapy has been growing in recent years. To meet the increasing clinical demand, LV production platforms will benefit from improved productivity and scalability to enable cost-effective manufacture of LV-based therapies. Here we report the adaptation of 293T cells to serum-free suspension cultures and the improvement of LV yields through transfection parameters optimization, process intensification and medium supplementation with nutrient boosters. Cells were sequentially adapted to different serum-free culture media, transfection parameters were optimized and the two best-performing conditions were selected to explore process intensification by increasing cell density at the time of transfection. LV production at higher cell densities increased volumetric titers up to 12-fold and lipid supplementation was the most efficient metabolic optimization strategy further enhancing LV productivity by 3-fold. Furthermore, cell concentration was identified and validated as an important source of transfection variability impairing cellular uptake of DNA polyplexes, impacting transfection efficiency and reducing LV titers down to 6-fold. This work contributes to improving LV-based gene therapy by establishing new scalable manufacturing platforms and providing key metabolic insights, unveiling important bioreaction parameters to improve vector yields.

Partial correction of immunodeficiency by lentiviral vector gene therapy in mouse models carrying Rag1 hypomorphic mutations.

Recombination activating genes (RAG) 1 and 2 defects are the most frequent form of severe combined immunodeficiency (SCID). Patients with residual RAG activity have a spectrum of clinical manifestations ranging from Omenn syndrome to delayed-onset combined immunodeficiency, often associated with granulomas and/or autoimmunity (CID-G/AI). Lentiviral vector (LV) gene therapy (GT) has been proposed as an alternative treatment to the standard hematopoietic stem cell transplant and a clinical trial for RAG1 SCID patients recently started. However, GT in patients with hypomorphic RAG mutations poses additional risks, because of the residual endogenous RAG1 expression and the general state of immune dysregulation and associated inflammation. In this study, we assessed the efficacy of GT in 2 hypomorphic Rag1 murine models (Rag1F971L/F971L and Rag1R972Q/R972Q), exploiting the same LV used in the clinical trial encoding RAG1 under control of the MND promoter. Starting 6 weeks after transplant, GT-treated mice showed a decrease in proportion of myeloid cells and a concomitant increase of B, T and total white blood cells. However, counts remained lower than in mice transplanted with WT Lin- cells. At euthanasia, we observed a general redistribution of immune subsets in tissues, with the appearance of mature recirculating B cells in the bone marrow. In the thymus, we demonstrated correction of the block at double negative stage, with a modest improvement in the cortical/medullary ratio. Analysis of antigenspecific IgM and IgG serum levels after in vivo challenge showed an amelioration of antibody responses, suggesting that the partial immune correction could confer a clinical benefit. Notably, no overt signs of autoimmunity were detected, with B-cell activating factor decreasing to normal levels and autoantibodies remaining stable after GT. On the other hand, thymic enlargement was frequently observed, although not due to vector integration and insertional mutagenesis. In conclusion, our work shows that GT could partially alleviate the combined immunodeficiency of hypomorphic RAG1 patients and that extensive efficacy and safety studies with alternative models are required before commencing RAG gene therapy in thesehighly complex patients.

Development and Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method for Sensitive Analysis of Residual Protein Tat Bh1-101 in Lentiviral Vectors for Gene Therapy.

Lentiviral (LV) vector-based gene therapy is gaining popularity for treating a wide range of diseases. Various LV vectors are being developed for transducing cells in cellular gene therapy at St. Jude. Some LV vectors are produced using stable 293T packaging cell lines, which includes gag-pol-rev-tat and virus-glycoprotein. Transactivating factor (transactivator of transcription [Tat]) is a regulatory protein that drastically increases the efficiency of lentiviral transcription. Residual analysis of Tat is critical for gene vector quality and safety. In this work, we developed a highly sensitive liquid chromatography-tandem mass spectrometry method for analysis of residual Tat in Lentivirus as an alternative to enzyme-linked immunosorbent assay. Residual Tat in LV can be accurately quantified with high specificity with a limit of detection of 0.3 ng/mL.

HIV Preintegration Transcription and Host Antagonism.

Retrovirus integration is an obligatory step for the viral life cycle, but large amounts of unintegrated DNA (uDNA) accumulate during retroviral infection. For simple retroviruses, in the absence of integration, viral genomes are epigenetically silenced in host cells. For complex retroviruses such as HIV, preintegration transcription has been found to occur at low levels from a large population of uDNA even in the presence of host epigenetic silencing mechanisms. HIV preintegration transcription has been suggested to be a normal early process of HIV infection that leads to the syntheses of all three classes of viral transcripts: multiply-spliced, singly-spliced, and unspliced genomic RNA; only viral early proteins such as Nef are selectively translated at low levels in blood CD4 T cells and macrophages, the primary targets of HIV. The initiation and persistence of HIV preintegration transcription have been suggested to rely on viral accessory proteins, particularly virion Vpr and de novo Tat generated from uDNA; both proteins have been shown to antagonize host epigenetic silencing of uDNA. In addition, stimulation of latently infected resting T cells and macrophages with cytokines, PKC activator, or histone deacetylase inhibitors has been found to greatly upregulate preintegration transcription, leading to low-level viral production or even replication from uDNA. Functionally, Nef synthesized from preintegration transcription is biologically active in modulating host immune functions, lowering the threshold of T cell activation, and downregulating surface CD4, CXCR4/CCR5, and HMC receptors. The early Tat activity from preintegration transcription antagonizes repressive minichromatin assembled onto uDNA. The study of HIV preintegration transcription is important to understanding virus-host interaction and antagonism, viral persistence, and the mechanism of integrase drug resistance. The application of unintegrated lentiviral vectors for gene therapy also offers a safety advantage for minimizing retroviral vector-mediated insertional mutagenesis.

Toward lentiviral vectors for antiangiogenic ocular gene therapy

Toward lentiviral vectors for antiangiogenic ocular gene therapy

CD90-targeted lentiviral vectors for HSC gene therapy

Hematopoietic stem cell (HSC) gene therapy is currently performed on CD34+ hematopoietic stem and progenitor cells containing less than 1% true HSCs and requiring a highly specialized infrastructure for cell manufacturing and transplantation. We have previously identified the CD34+CD90+ subset to be exclusively responsible for short- and long-term engraftment. However, purification and enrichment of this subset is laborious and expensive. HSC-specific delivery agents for the direct modification of rare HSCs are currently lacking. Here, we developed novel targeted viral vectors to specifically transduce CD90-expressing HSCs. Anti-CD90 single chain variable fragments (scFvs) were engineered onto measles- and VSV-G-pseudotyped lentiviral vectors that were knocked out for native targeting. We further developed a custom hydrodynamic titration methodology to assess the loading of surface-engineered capsids, measure antigen recognition of the scFv, and predict the performance on cells. Engineered vectors formed with minimal impairment in the functional titer, maintained their ability to fuse with the target cells, and showed highly specific recognition of CD90 on cells exvivo. Most important, targeted vectors selectively transduced human HSCs with secondary colony-forming potential. Our novel HSC-targeted viral vectors have the potential to significantly enhance the feasibility of exvivo gene therapy and pave the way for future invivo applications.

Novel lentiviral vectors for gene therapy of sickle cell disease combining gene addition and gene silencing strategies

Sickle cell disease (SCD) is due to a mutation in the β-globin gene causing production of the toxic sickle hemoglobin (HbS; α2βS 2). Transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) transduced with lentiviral vectors (LVs) expressing an anti-sickling β-globin (βAS) is a promising treatment; however, it is only partially effective, and patients still present elevated HbS levels. Here, we developed a bifunctional LV expressing βAS3-globin and an artificial microRNA (amiRNA) specifically downregulating βS-globin expression with the aim of reducing HbS levels and favoring βAS3 incorporation into Hb tetramers. Efficient transduction of SCD HSPCs by the bifunctional LV led to a substantial decrease of βS-globin transcripts in HSPC-derived erythroid cells, a significant reduction of HbS+ red cells, and effective correction of thesickling phenotype, outperforming βAS gene addition and BCL11A gene silencing strategies. The bifunctional LV showed a standard integration profile, and neither HSPC viability, engraftment, and multilineage differentiation nor the erythroid transcriptome and miRNAome were affected by the treatment, confirming the safety of this therapeutic strategy. In conclusion, the combination of gene addition and gene silencing strategies can improve the efficacy of current LV-based therapeutic approaches without increasing the mutagenic vector load, thus representing a novel treatment for SCD.

Improved Lentiviral In Vivo Gene Therapy for Chronic Granulomatous Disease

Background: Chronic granulomatous disease (CGD) is a congenital immunodeficiency characterized by severe life-threatening infections. CGD patients lack reactive oxygen species (ROS) in phagocytic leukocytes and often develop widespread tissue granulomas. The current treatment for CGD is lifelong antibiotic prophylaxis or hematopoietic stem cell transplantation (HSCT). However, most patients cannot find matched donors, and HSCT is known to have increased risk in CGD patients. Gene therapy has become a promising treatment for CGD. At present, "ex vivo" HSC gene therapy is the widely applied strategy. "In vivo" gene therapy is based on direct delivery of gene therapy vectors into patients, which could overcome the difficulties in HSCT. Methods: We have developed an in vivo CGD gene therapy strategy by intravenous (iv) injection of lentiviral vectors (LVs) carrying an universal EF1a promoter or a myeloid-specific miR223 promoter driving the expression of CYBB (cytochrome B-245 beta chain, or p91-phox) in X-CGD mice (X-CGD; B6.129S-Cybbtm1Din/J). Each mouse received 1x109 transduction units (TU) of LV-CYBB via iv injection after non-myeloablative conditioning. A second injection of 1x109 TU of LV-CYBB after re-conditioning was given 12 weeks later. Results: We developed a chemotherapy conditioning regimen to improve gene delivery efficiency by using busulfan, cyclophosphamide and dexamethasone. The X-CGD mice received two iv injections of LVs, and the expression and function of the LV-CYBB gene were analyzed by intracellular gp91-phox staining and DHR123 assay 4 weeks after each injection. Based on flow cytometry, we detected gp91-phox positive cells in the blood as the following, EF1a: 1.14-5.18% and 5.27-14.36%, and miR223: 1.65-5.17% and 4.59-17.89% after the first and the second injections, respectively, as compared with the WT mice: 79.9-87.6%. Functional analyses of ROS showed the following, EF1a: 1.93-5.2% and 2.39-18.51%, miR223: 2.65-6.67% and 3.75-19.8%, after the first and the second injections respectively, as compared with WT: 72.6-85.31% (Figure A). The LV vector copy number (VCN) in the peripheral blood mononuclear cells was detected by qPCR as the following, EF1a: 1.11-2.32% and 1.35-9.32%, and miR223: 1.01-2.39% and 2.71-8.51% after the first and the second injections, respectively. These results indicated that iv LV injection effectively delivered and expressed the LV-CYBB gene in vivo, and repeated injection of the LVs could increase the gene transfer efficiency in the CGD mice. Importantly, after the second injection, we found no antibody response to the LV particle-associated protein p24. However, after 3-12 months, markedly reduced VCN was found in the blood of the treated mice, suggesting exhaustion of the transgenes in the somatic cells. To improve the gene delivery into HSCs, we treated the mice with AMD3100 to mobilize HSCs into peripheral blood. The mice were treated with chemo-conditioning and AMD3100 prior to LV ivinjection. After four weeks, the gp91-phox positive HSCs of total Sca1+ HSCs in blood with or without (+/-) AMD3100 treatment were examined by flow cytometry, and the results showed the following, EF1a: (-) 9.52-15.43%, (+) 16.1-29.4%, and miR223: (-) 4.49-10.35% and (+) 7.06-16.84%. To confirm stem cell gene transfer, we transplanted the bone marrow cells from the LV-CYBB-treated CGD mice into untreated CGD mice. The VCN in blood was examined before (donor mice) and 4 weeks after (recipient mice) the HSCT, and the results showed the following, EF1a: before 4.49% (in the donor) and after 1.05-2.57% (in the recipients), and miR223: before 4.91% (in the donor) and after 1.04-2.69% (in the recipients) (Figure B). This transplantation study confirmed that the AMD3100 treatment effectively increased iv LV gene delivery into HSCs. Conclusion: We have developed an improved LV iv gene delivery strategy by applying chemo-conditioning and AMD3100 mobilization prior to the injection of the LVs into CGD mice. This in vivo LV gene therapy strategy could potentially be translated into future CGD gene therapy applications. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

In vivo lentiviral vector gene therapy to cure hereditary tyrosinemia type 1 and prevent development of precancerous and cancerous lesions

Conventional therapy for hereditary tyrosinemia type-1 (HT1) with 2-(2-nitro-4-trifluoromethylbenzoyl)−1,3-cyclohexanedione (NTBC) delays and in some cases fails to prevent disease progression to liver fibrosis, liver failure, and activation of tumorigenic pathways. Here we demonstrate cure of HT1 by direct, in vivo administration of a therapeutic lentiviral vector targeting the expression of a human fumarylacetoacetate hydrolase (FAH) transgene in the porcine model of HT1. This therapy is well tolerated and provides stable long-term expression of FAH in pigs with HT1. Genomic integration displays a benign profile, with subsequent fibrosis and tumorigenicity gene expression patterns similar to wild-type animals as compared to NTBC-treated or diseased untreated animals. Indeed, the phenotypic and genomic data following in vivo lentiviral vector administration demonstrate comparative superiority over other therapies including ex vivo cell therapy and therefore support clinical application of this approach.

Lentiviral Vectors for Ocular Gene Therapy.

This review offers the basics of lentiviral vector technologies, their advantages and pitfalls, and an overview of their use in the field of ophthalmology. First, the description of the global challenges encountered to develop safe and efficient lentiviral recombinant vectors for clinical application is provided. The risks and the measures taken to minimize secondary effects as well as new strategies using these vectors are also discussed. This review then focuses on lentiviral vectors specifically designed for ocular therapy and goes over preclinical and clinical studies describing their safety and efficacy. A therapeutic approach using lentiviral vector-mediated gene therapy is currently being developed for many ocular diseases, e.g., aged-related macular degeneration, retinopathy of prematurity, inherited retinal dystrophies (Leber congenital amaurosis type 2, Stargardt disease, Usher syndrome), glaucoma, and corneal fibrosis or engraftment rejection. In summary, this review shows how lentiviral vectors offer an interesting alternative for gene therapy in all ocular compartments.

Characterization of an immune-evading doxycycline-inducible lentiviral vector for gene therapy in the spinal cord

Gene therapy is a powerful approach to promote spinal cord regeneration. For a clinical application it is important to restrict therapeutic gene expression to the appropriate time window to limit unwanted side effects. The doxycycline (dox)-inducible system is a widely used regulatable gene expression platform, however, this system depends on a bacterial-derived immunogenic transactivator. The foreign origin of this transactivator prevents reliable regulation of therapeutic gene expression and currently limits clinical translation. The glycine-alanine repeat (GAR) of Epstein-Barr virus nuclear antigen-1 protein inhibits its presentation to cytotoxic T cells, allowing virus-infected cells to evade the host immune system. We developed a chimeric transactivator (GARrtTA) and show that GARrtTA has an immune-evading advantage over “classical” rtTA in vivo. Direct comparison of lentiviral vectors expressing rtTA and GARrtTA in the rat spinal cord shows that the GARrtTA system is inducible for 6 doxycycline-cycles over a 47 week period, whereas with the rtTA-based system luciferase reporter expression declines during the 3rd cycle and is no longer re-inducible, indicating that GARrtTA provides an immune-advantage over rtTA. Immunohistochemistry revealed that GARrtTA expressing cells in the spinal cord appear healthier and survive better than rtTA expressing cells. Characterization of the immune response shows that expression of GARrtTA, in contrast to rtTA, does not recruit cytotoxic T-cells to the transduced spinal cord. This study demonstrates that fusion of the GAR domain to rtTA results in a functional doxycycline-inducible transactivator with a clear immune-advantage over the classical rtTA in vivo.

A Novel RPS19-Edited Hematopoietic Stem Cell Model of Diamond-Blackfan Anemia for Development of Lentiviral Vector Gene Therapy

Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia that typically manifests in infancy as macrocytic anemia with reticulocytopenia. About 80% of DBA cases are caused by heterozygous loss-of-function mutations or deletions in one of 23 ribosomal proteins (RP) genes, with RPS19 being affected in ~25% of patients. Current therapies are suboptimal, and it is difficult to obtain DBA patient hematopoietic stem and progenitor cells (HSPCs) in sufficient quantity for preclinical development of new therapies. To address this gap, we used CRISPR/Cas9-edited healthy donor CD34 + HSPCs to create a novel model of RPS19-mutated DBA and used this model to develop an efficient RPS19-encoding lentiviral vector (LV) for gene therapy.Healthy donor CD34 + HSPCs were electroporated with ribonucleoprotein (RNP) complex consisting of Cas9 and guide RNAs (gRNAs) targeting RPS19 or the AAVS1 locus as a negative control, then grown in medium to support erythroid differentiation. All gRNAs analyzed generated high-frequency on-target insertion-deletion (indels) mutations (Fig. A). RPS19 indels specifically declined over time, suggesting that RPS19 disruption impairs cell proliferation and/or survival. To rescue the defect, we constructed a third-generation, self-inactivating LV expressing RPS19+GFP (RPS19/GFP LV). Transduction was optimized using poloxamer and prostaglandin E2 and linear transduction efficiency was noted even at high multiplicity of infection (MOI) (Fig. B). An MOI of 20 was used for subsequent experiments. In methylcellulose medium, RPS19 RNP-treated cells generated 72% fewer burst forming unit-erythroid (BFU-E) colonies compared to AAVS1 RNP-treated control cells. RPS19/GFP LVs with three different promoters (EF1α short, EF1α long and MND) partially restored BFU-E formation similarly (Fig. C); the EF1α short promoter was chosen for subsequent experiments due its track record for clinical use. We down-titrated the RNP concentration to generate a total indel frequency of ~25%, which resulted in approximately equal frequencies of RPS19 +/+ and RPS19 +/- BFU-E colonies. RPS19 -/- colonies were detected only after edited HSPCs were rescued by RPS19/GFP LV, due to lethality of this genotype (Fig. D). Transfection of CD34 + HSPCs with RPS19 RNP caused a 49% reduction in cell number after 14 days of liquid culture in erythroid differentiation medium compared to control HSPCs; this was corrected by treatment with RPS19/GFP LV (Fig. E). RPS19 RNP treatment of CD34 + HSPCs had no effect on the expansion of cells grown under myeloid differentiation conditions (Fig. F).We analyzed RNP-treated CD34 + cells further by transducing them with RPS19/GFP LV or control LV encoding GFPalone, transplanting them into immunodeficient NSGW mice and analyzing human donor cell progeny in mouse bone marrow after 16 weeks. In cells treated with AAVS1 RNP and GFP LV, the indel frequency dropped from 27.2±1.5% (SD) at 72 hours after editing (input) to 15.5±4.4% at 16 weeks post-transplant (43% reduction) (Fig. G). In HSPCs treated with RPS19 RNP and GFP LV the indel frequency dropped from 20.9±3.1% in input cells to 1.8±0.9% after 16 weeks (Fig. G) (92% reduction). In contrast, the indel frequency of donor HSPCs treated with RPS19 RNP and RPS19/GFP LV dropped from 23.6±2.7% in input cells to 8.4±1.6% (64% reduction), which represents a 5-fold increase in indel frequency compared to treatment with control GFP LV (p< 0.01)(Fig. G). In flow cytometry-purified, donor HSPC-derived myeloid, B-lymphocyte, HSPC and erythroid lineages at 16 weeks after xenotransplantation, the mean indel rates were 1.3% to 2.5% in cells derived from HSPCs treated with RPS19 RNP and GFP LV. Indel rates ranged from 6.9% to 9.2% in the progeny of input HSPCs that were rescued by RPS19/GFP LV, representing a 4-6-fold increase compared to transduction with control GFP LV (p<0.01) (Fig. H).In summary, our studies show that Cas9-mediated disruption of RPS19 in CD34 + HSPCs causes a selective erythroid defect in RPS19 +/- cells, recapitulating the canonical DBA defect. Additionally, deficient bone marrow repopulation by RPS19 +/- cells suggests an HSC defect, consistent with pancytopenia that is observed in many older DBA patients. The optimized RPS19 LV transduces HSPCs at high efficiency and alleviates both defects, supporting its potential utility for DBA therapy. [Display omitted] DisclosuresYen: Beam Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Weiss: Beam Therapeutics: Current holder of stock options in a privately-held company; Forma Therapeutics: Consultancy; Novartis: Consultancy; Cellarity Inc.: Consultancy.

Intravenous Lentiviral Gene Therapy for Hemophilia a: In Vitro and In Vivo Assessment of Universal Versus Tissue-Specific Promoters

Background：Hemophilia A (HA) is an X-linked monogenic coagulation disorder resulting from deficiency of the factor VIII (FVIII, F8) gene in the intrinsic coagulation cascade. The current treatment of HA is based on protein replacement therapy (PRT) through plasma-derived coagulation factors or recombinant proteins with limitations of short half-life, high cost, and life-time requirement of the treatment. Gene therapy has become a promising treatment for HA.Methods and Materials: We developed an advanced lentiviral vector (LV) system for intravenous (iv) F8 gene therapy. A selective codon optimized and B-domain deleted human F8 (hF8BDD) gene was synthesized, sequenced and functionally verified. LVs carrying a universal EF1α promoter, or several modified tissue-specific promoters including endothelial- (VEC), endothelial and epithelial- (KDR), and two megakaryocyte-specific (ITGA and Gp) promoters, were biologically and immunologically characterized in vitro using human endothelial and megakaryocytic cell lines, EA-hy926 and DAMI, and in vivo using F8 knockout (KO) mice.Results: We investigated the different LV promoter constructs and found that LV-VEC-F8BDD exhibited the highest virus packaging (1.3x10 9 transduction units/ml) and transduction efficiencies compared with the other LV constructs. Tissue-specific expression of the VEC, KDR, ITGA and Gp promoters was confirmed in EA-hy926 and DAMI cells by RT-PCR, Western blot and ELISA analyses. We detected F8 activities close to 6 folds and 4.5 folds above the normal plasma level from the EF1a LV-F8BDD transduced EA-hy926 cells and DAMI cells, respectively, whereas the VEC LV in EA-hy926 cells and the ITGA-LV in DAMI cells exhibited F8 activities at 1.5 folds and 5 folds above the normal plasma level, respectively. In vivo studies in F8 KO mice via iv injection of LVs after reduced radiation conditioning illustrated preferential vector expression in different cell lineages, with high expression of EF1a vector in CD11b, F4/80 and Ly-6G positive immune cells, and preferential expression of VEC vector in CD31 positive endothelial cells, and ITGA and Gp vectors in CD41 positive megakaryocytes. In addition, we detected variable phenotypic corrections as well as anti-F8 immune responses in the F8 KO mice treated with the different LVs. The iv deliveries of VEC and Gp F8BDD vectors illustrated therapeutic F8 activities over time, around 25% and 8%, respectively, in 60 days, which increased to high levels (80% and 25%, respectively) after 120 days (Figure A). Kinetic analyses of anti-F8 IgG and inhibitor titers (Bethesda assay) of the treated mice showed that the VEC vector exhibited the lowest F8 inhibitory immune response over time (Figure B).Conclusion: Based on the in vitro and in vivo studies, our results suggest that for HA gene therapy, optimal rather than high F8 expression is critical, and tissue-specific expression but not universal expression can reduce adverse inhibitor effect. We demonstrated that the LV-VEC-F8BDD vector displayed high tissue specificity in vivo, and high transgene delivery efficiency, high coagulation function and low immunogenicity. In addition, iv LV gene therapy could be a safe, convenient and effective HA gene therapy strategy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Designing Lentiviral Vectors for Gene Therapy of Genetic Diseases.

Lentiviral vectors are the most frequently used tool to stably transfer and express genes in the context of gene therapy for monogenic diseases. The vast majority of clinical applications involves an ex vivo modality whereby lentiviral vectors are used to transduce autologous somatic cells, obtained from patients and re-delivered to patients after transduction. Examples are hematopoietic stem cells used in gene therapy for hematological or neurometabolic diseases or T cells for immunotherapy of cancer. We review the design and use of lentiviral vectors in gene therapy of monogenic diseases, with a focus on controlling gene expression by transcriptional or post-transcriptional mechanisms in the context of vectors that have already entered a clinical development phase.

Preclinical studies of efficacy thresholds and tolerability of a clinically ready lentiviral vector for pyruvate kinase deficiency treatment

Pyruvate kinase deficiency (PKD) is a rare autosomal recessive disorder caused by mutations in the PKLR gene. PKD is characterized by non-spherocytic hemolytic anemia of variable severity and may be fatal in some cases during early childhood. Although not considered the standard of care, allogeneic stem cell transplantation has been shown as a potentially curative treatment, limited by donor availability, toxicity, and incomplete engraftment. Preclinical studies were conducted to define conditions to enable consistent therapeutic reversal, which were based on our previous data on lentiviral gene therapy for PKD. Improvement of erythroid parameters was identified by the presence of 20%–30% healthy donor cells. A minimum vector copy number (VCN) of 0.2−0.3 was required to correct PKD when corrected cells were transplanted in a mouse model for PKD. Biodistribution and pharmacokinetics studies, with the aim of conducting a global gene therapy clinical trial for PKD patients (RP-L301-0119), demonstrated that genetically corrected cells do not confer additional side effects. Moreover, a clinically compatible transduction protocol with mobilized peripheral blood CD34+ cells was optimized, thus facilitating the efficient transduction on human cells capable of repopulating the hematopoiesis of immunodeficient mice. We established conditions for a curative lentiviral vector gene therapy protocol for PKD.

Recent advances in lentiviral vectors for gene therapy.

Lentiviral vectors (LVs), derived from human immunodeficiency virus, are powerful tools for modifying the genes of eukaryotic cells such as hematopoietic stem cells and neural cells. With the extensive and in-depth studies on this gene therapy vehicle over the past two decades, LVs have been widely used in both research and clinical trials. For instance, third-generation and self-inactive LVs have been used to introduce a gene with therapeutic potential into the host genome and achieve targeted delivery into specific tissue. When LVs are employed in leukemia, the transduced T cells recognize and kill the tumor B cells; in β-thalassemia, the transduced CD34+ cells express normal β-globin; in adenosine deaminase-deficient severe combined immunodeficiency, the autologous CD34+ cells express adenosine deaminase and realize immune reconstitution. Overall, LVs can perform significant roles in the treatment of primary immunodeficiency diseases, hemoglobinopathies, B cell leukemia, and neurodegenerative diseases. In this review, we discuss the recent developments and therapeutic applications of LVs. The safe and efficient LVs show great promise as a tool for human gene therapy.

Size-Controlled and Shelf-Stable DNA Particles for Production of Lentiviral Vectors.

Polyelectrolyte complex particles assembled from plasmid DNA (pDNA) and poly(ethylenimine) (PEI) have been widely used to produce lentiviral vectors (LVVs) for gene therapy. The current batch-mode preparation for pDNA/PEI particles presents limited reproducibility in large-scale LVV manufacturing processes, leading to challenges in tightly controlling particle stability, transfection outcomes, and LVV production yield. Here we identified the size of pDNA/PEI particles as a key determinant for a high transfection efficiency with an optimal size of 400–500 nm, due to a cellular-uptake-related mechanism. We developed a kinetics-based approach to assemble size-controlled and shelf-stable particles using preassembled nanoparticles as building blocks and demonstrated production scalability on a scale of at least 100 mL. The preservation of colloidal stability and transfection efficiency was benchmarked against particles generated using an industry standard protocol. This particle manufacturing method effectively streamlines the viral manufacturing process and improves the production quality and consistency.