Lentivirus-based Gene Therapy Research Articles

Systematic Mapping of Gene-Editable Mutations in GATA2 and SAMD9/SAMD9L Syndromes

Germline predisposition to bone marrow failure (BMF) and myelodysplastic syndromes (MDS) cause substantial economic and psychosocial burden and early mortality in affected individuals. Due to the limitations and risks of hematopoietic stem cell transplantation, there is an urgent need for new and innovative treatment approaches. Conventional lentiviral-based gene therapy is not feasible for most BMF/MDS predisposing genes because their uncontrolled overexpression is detrimental and lentiviral vectors might cause insertional mutagenesis. However, new technologies that precisely modify the endogenous allele can overcome these limitations. Base editing (BE) using cytosine base editors (CBE) and adenine base editors (ABE) as well as prime editing (PE) are advanced gene editing techniques with great therapeutic promise. The goal of this project is to develop a comprehensive map of gene-editable mutations in the GATA2, SAMD9, and SAMD9L genes as a preclinical effort to facilitate gene editing trials. Germline heterozygous mutations in these key predisposing genes lead to highly penetrant disorders characterized by progressive immunodeficiency and BMF/MDS risk, with particularly high prevalence in children, as recently reported by us (Sahoo, Nature Medicine 2021) and others. For in depth analysis, we focused on the 5 most recurrent mutations in respective genes and devise strategies to correct mutations in cellular models. We first created a comprehensive database encompassing reported and unpublished pathogenic mutations (single nucleotide variants [SNVs] and <20bp insertion/deletions[indels]). In total, we annotated 506 cases with GATA2, 195 cases with SAMD9, and 187 cases with SAMD9L mutations. SNVs were detected in 83.2% (421/506), 94.4% (184/195), and 93.1% (174/187) patients with GATA2, SAMD9, SAMD9L mutations, respectively, while remaining cases had indels. Among all GATA2 cases, 59.9% (303/506) mutations are editable by ABE, 4.5% (23/506) by CBE, while remaining 35.6% (180/506) can be “rescued” by PE ( Fig. 1A). For SAMD9, 34.4% (67/195) cases were found to be amenable to editing by ABE, 30.3% (59/195) by CBE, and 35.4% (69/195) using PE. For SAMD9L, 47.1% (88/187) are editable by ABE, 20.3% (38/187) by CBE, and 32.6% (61/187) using PE. Having a well-annotated mutation list, we proceeded to identify the top 5 most common mutations in each gene ( Fig. 1B). Remarkably, all 5 mutations in both GATA2 and SAMD9 and 4 out of 5 top mutations in SAMD9L and amenable to BE. Next, we explored in depth the target editing window, protospacer adjacent motif (PAM) sites, and bystander editing effect (utilizing BE that recognize the canonical [NGG], and non-canonical PAM sequences [NG and near PAMless SpRY-NRN/NYN]. This stepwise approach resulted in successful identification of at least one potential guide RNA for each mutation with predicted low bystander editing. Ongoing work includes guide RNA validation, off-target analysis, and assessment of gene-editing efficiency using model cell lines. In conclusion, we have undertaken a systematic analysis of the gene editing landscape for frequent germline driver mutations in BMF/MDS. Our findings reveal promising editing strategies for the majority of prevalent mutations in key predisposing genes GATA2, SAMD9 and SAMD9L using CBE, ABE, or PE editors, and lay a groundwork to enable future clinical translation.

Lentiviral delivered aflibercept OXB-203 for treatment of neovascular AMD

Neovascular age-related macular degeneration (nAMD) is a leading cause of blindness in the aging population, with vascular endothelial growth factor (VEGF) playing a key role. Treatment with recombinant anti-VEGFs is the current standard of care; however, it is only effective for 1-2months at a time and requires re-administration. Gene therapy could pave the way for stable, long-term expression of therapeutic anti-VEGF with a single dose, reducing the frequency of treatment and potentially improving clinical outcomes. As such, we have developed OXB-203, a lentiviral-based gene therapy encoding the anti-VEGF protein aflibercept. Aflibercept derived from OXB-203 exhibited comparable invitro binding characteristics to VEGF as recombinant aflibercept. Furthermore, its biological potency was demonstrated by the equivalent inhibition of VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and tubule formation as recombinant aflibercept. In a rat choroidal neovascularization (CNV) model of nAMD, a single subretinal administration of OXB-203 reduced laser-induced CNV lesion areas analogous to an intravitreal bolus of recombinant aflibercept. Finally, in a head-to-head comparative study, aflibercept derived from OXB-203 was shown to be expressed at significantly higher levels in ocular tissues than from an AAV8-aflibercept vector following a single subretinal delivery to rats. These findings support the therapeutic potential of OXB-203 for the management of nAMD.

Lentiviral gene therapy reverts GPIX expression and phenotype in Bernard-Soulier syndrome type C

Bernard-Soulier syndrome (BSS) is a rare congenital disease characterized by macrothrombocytopenia and frequent bleeding. It is caused by pathogenic variants in three genes (GP1BA, GP1BB, or GP9) that encode for the GPIbα, GPIbβ, and GPIX subunits of the GPIb-V-IX complex, the main platelet surface receptor for von Willebrand factor, being essential for platelet adhesion and aggregation. According to the affected gene, we distinguish BSS type A1 (GP1BA), type B (GP1BB), or type C (GP9). Pathogenic variants in these genes cause absent, incomplete, or dysfunctional GPIb-V-IX receptor and, consequently, a hemorrhagic phenotype. Using gene-editing tools, we generated knockout (KO) human cellular models that helped us to better understand GPIb-V-IX complex assembly. Furthermore, we developed novel lentiviral vectors capable of correcting GPIX expression, localization, and functionality in human GP9-KO megakaryoblastic cell lines. Generated GP9-KO induced pluripotent stem cells produced platelets that recapitulated the BSS phenotype: absence of GPIX on the membrane surface and large size. Importantly, gene therapy tools reverted both characteristics. Finally, hematopoietic stem cells from two unrelated BSS type C patients were transduced with the gene therapy vectors and differentiated to produce GPIX-expressing megakaryocytes and platelets with a reduced size. These results demonstrate the potential of lentiviral-based gene therapy to rescue BSS type C.

Severe hematopoietic stem cell inflammation compromises chronic granulomatous disease gene therapy

X-linked chronic granulomatous disease (CGD) is associated with defective phagocytosis, life-threatening infections, and inflammatory complications. We performed a clinical trial of lentivirus-based gene therapy in four patients (NCT02757911). Two patients show stable engraftment and clinical benefits, whereas the other two have progressively lost gene-corrected cells. Single-cell transcriptomic analysis reveals a significantly lower frequency of hematopoietic stem cells (HSCs) in CGD patients, especially in the two patients with defective engraftment. These two present a profound change in HSC status, a high interferon score, and elevated myeloid progenitor frequency. We use elastic-net logistic regression to identify a set of 51 interferon genes and transcription factors that predict the failure of HSC engraftment. In one patient, an aberrant HSC state with elevated CEBPβ expression drives HSC exhaustion, as demonstrated by low repopulation in a xenotransplantation model. Targeted treatments to protect HSCs, coupled to targeted gene expression screening, might improve clinical outcomes in CGD.

Characterization of a new IN-I-PpoI fusion protein and a homology-arm containing transgene cassette that improve transgene expression persistence and 28S rRNA gene-targeted insertion of lentiviral vectors.

Targeting transgene integration into a safe genomic locus would be very important for gene therapy. We have generated lentivirus vectors containing the ribosomal RNA-recognising I-PpoI endonuclease fused to viral integrase, and transgene cassettes with target site homology arms to enhance insertion targeting. These new vectors were characterised with respect to the persistence of transgene expression, insertion targeting efficiency and chromosomal integrity of the transduced cells. The aim was to find an optimally safe and effective vector for human gene therapy. Fusion protein vectors with high endonuclease activity were the most effective in the accurate targeting of transgene insertion. The homology construct increased the insertion targeting efficiency to 28% in MRC-5 cells. However, karyotyping analysis showed that the high endonuclease activity induced the formation of derivative chromosomes in as many as 24% of the analysed primary T lymphocytes. The persistence of transgene expression was excellent in homology arm-containing fusion protein vectors with reduced endonuclease activity, and these fusion proteins did not cause any detectable chromosomal rearrangements attributable to the endonuclease activity. We thus conclude that instead of the fusion protein vectors that carry a highly active endonuclease, our vectors with the ability to tether the lentivirus preintegration complex to benign loci in the genome without high ribosomal DNA cleavage activity are better suited for lentivirus-based gene therapy applications.

Knowledge to date on secondary malignancy following hematopoietic cell transplantation for sickle cell disease

Allogeneic hematopoietic cell transplantation, gene therapy, and gene editing offer a potential cure for sickle cell disease (SCD). Unfortunately, myelodysplastic syndrome and acute myeloid leukemia development have been higher than expected after graft rejection following nonmyeloablative conditioning and lentivirus-based gene therapy employing myeloablative busulfan for SCD. Somatic mutations discovered in 2 of 76 patients who rejected their grafts were identified at baseline at much lower levels. While a whole-genome sequencing analysis reported no difference between patients with SCD and controls, a study including whole-exome sequencing revealed a higher prevalence of clonal hematopoiesis in individuals with SCD compared with controls. Genetic risk factors for myeloid malignancy development after curative therapy for SCD are currently being explored. Once discovered, decisions could be made about whether gene therapy may be feasible vs allogeneic hematopoietic cell transplant, which results in full donor chimerism. In the meantime, care should be taken to perform a benefit/risk assessment to help patients identify the best curative approach for them. Long-term follow-up is necessary to monitor for myeloid malignancies and other adverse effects of curative therapies for SCD.

Adrenoleukodystrophy Protein (ALDP) Antibody Formation and Graft Loss in Gene Therapy for Adrenoleukodystrophy

A nine-year boy with adrenoleukodystrophy (ALD) due to an ABCD1 whole gene deletion was diagnosed with the active cerebral form of disease characterized by demyelination and gadolinium enhancement on brain MRI. Allogeneic hematopoietic stem cell transplant (HCT) or lentiviral correction of autologous CD34+cell-based gene therapy (GT) are the only known mechanisms to arrest the progression of cerebral disease. He was treated with GCSF/plerixafor to mobilize autologous CD34+ cells which were then transduced with the ABCD1 expressing lentiviral vector. He received myeloablative conditioning with busulfan and fludarabine followed by lentivirus-based GT. Initial viral copy number (VCN) was 0.8592 in whole blood at 30 days post HCT and MRI's showed disease stabilization with significant decrease in gadolinium enhancement at 60 and 100 days after GT. Unfortunately, VCN then declined and subsequent MRI's showed re-emergence of gadolinium enhancement and disease progression at 6 months after GT. Antibodies to the ABCD1 gene product (ALDP) were detected in the patient's plasma by immunoblot at 9 months after GT. Antisera also showed specific reactivity to human peroxisomes in immunofluorescence assays, suggesting that generation of an immune response had occurred following GT. Evaluation of pre-GT plasma did not find any anti-ALDP. Evaluation of post HCT plasma from other patients undergoing either GT or allo-HCT did not show anti-ALPD reactivity suggesting this was a unique phenomenon to this patient. Peptide mapping indicated the antisera bound mostly to the C-terminal portion of ALDP in five distinct peptide regions. Subtyping of the antibody indicated it was IgG and not IgM at 9 months post GT. Specific anti-ALDP IgG Subtypes detected were IgG1 and IgG3. Immune adjuvant therapy using rituximab and daratumumab failed to significantly reduce plasma reactivity to ALDP. Due to disease advancement on MRI, an allogeneic HCT was performed approximately 12 months after initial GT with an 5/6 HLA matched umbilical cord blood unit. Donor neutrophil engraft occurred on day +17, donor platelet engraftment occurred on day +35. Chimerism studies revealed 100% engraftment in CD33+cells and 90% engraftment in CD3+ cells by 60 days post HCT. The MRI's at 30-, 60-, and 100-days post allo-HCT showed stabilizing cerebral disease and fading gadolinium enhancement. Importantly, plasma collected at 30-, 60-days, and 100-days post allo-HCT showed no reactivity to ALPD. Conclusion. This is the first case of anti-ALDP antibodies occurring after gene therapy for cerebral ALD. Antibody generation may have been related to the unique ABCD1 whole gene deletion condition within this child which is very rare as the majority of ALD patients have missense or non-sense mutations in ABCD1. Whether anti-ALDP presence was a causal or an unrelated phenomenon to the loss of the autologous vector-containing cells is not currently known at the time of submission. These findings highlight to need to scrutinize patients prior to before and after GT for the presence of antibody to the protein product being introduced. This may be especially important in the case where the patient's immune system has never seen the protein (like in this case of a whole gene deletion) and therefore would have never been "educated".

3’ UTR-truncated HMGA2 overexpression induces non-malignant in vivo expansion of hematopoietic stem cells in non-human primates

Vector-mediated mutagenesis remains a major safety concern for many gene therapy clinical protocols. Indeed, lentiviral-based gene therapy treatments of hematologic disease can result in oligoclonal blood reconstitution in the transduced cell graft. Specifically, clonal expansion of hematopoietic stem cells (HSCs) highly expressing HMGA2, a chromatin architectural factor found in many human cancers, is reported in patients undergoing gene therapy for hematologic diseases, raising concerns about the safety of these integrations. Here, we show for the first time in vivo multilineage and multiclonal expansion of non-human primate HSCs expressing a 3’ UTR-truncated version of HMGA2 without evidence of any hematologic malignancy >7 years post-transplantation, which is significantly longer than most non-human gene therapy pre-clinical studies. This expansion is accompanied by an increase in HSC survival, cell cycle activation of downstream progenitors, and changes in gene expression led by the upregulation of IGF2BP2, a mRNA binding regulator of survival and proliferation. Thus, we conclude that prolonged ectopic expression of HMGA2 in hematopoietic progenitors is not sufficient to drive hematologic malignancy and is not an acute safety concern in lentiviral-based gene therapy clinical protocols.

Human Lentiviral Gene Therapy Restores the Cellular Phenotype of Autosomal Recessive Complete IFN-γR1 Deficiency

Autosomal recessive (AR) complete interferon-γ receptor 1 (IFN-γR1) deficiency, also known as one genetic etiology of Mendelian susceptibility to mycobacterial disease (MSMD), is a life-threatening congenital disease leading to premature death. Affected patients present a pathognomonic predisposition to recurrent and severe infections with environmental mycobacteria or the Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine. Current therapeutic options are limited to antibiotic treatment and hematopoietic stem cell transplantation, however with poor outcome. Given the clinical success of gene therapy, we introduce the first lentiviral-based gene therapy approach to restore expression and function of the human IFN-γR-downstream signaling cascade. In our study, we developed lentiviral vectors constitutively expressing the human IFN-γR1 and demonstrate stable transgene expression without interference with cell viability and proliferation in transduced human hematopoietic cells. Using an IFN-γR1-deficient HeLa cell model, we show stable receptor reconstitution and restored IFN-γR1 signaling without adverse effect on cell functionality. Transduction of both SV40-immortalized and primary fibroblasts derived from IFN-γR1-deficient MSMD patients was able to recover IFN-γR1 expression and restore type II IFN signaling upon stimulation with IFN-γ. In summary, we highlight lentiviral vectors to correct the IFN-γ mediated immunity and present the first gene therapy approach for patients suffering from AR complete IFN-γR1 deficiency.

Gene therapy for PNP deficiency protocol

Purine nucleoside phosphorylase (PNP) is a key enzyme required for the degradation of purine nucleosides into uric acid or their salvage into nucleic acids. Patients who are deficient in PNP suffer from progressive T cell immunodeficiency, with increased susceptibility to infections, autoimmunity, and neurologic abnormalities. In the absence of successful treatment to restore immune function, these patients rarely survive to adulthood. Hematopoietic stem cell transplantation is the only known cure for PNP deficiency. Use of an HLA-matched donor is preferable as the outcome with alternative donors have been variable; however, this option is rarely available. Gene therapy represents a therapeutic option that bypasses the need for a donor, and thus associated complications. Although first generation γ-retroviral vectors have been successful in some immunodeficiencies, in others, evidence of insertional mutagenesis prompted a halt in their use. More recently, the introduction of safer lentiviral vectors holds promise in offering a viable option to treat immunodeficiency. Here, we present a clinical trial protocol utilizing self-inactivating lentiviral vectors to treat PNP deficiency. Patients will be evaluated up to 3 years post-transplantation to determine the safety of lentiviral-treated stem cell infusion, as well as the extent of immune reconstitution. Statement of novelty: This protocol describes the novel treatment of PNP deficiency using lentiviral-based gene therapy.

Development of the First World Health Organization Lentiviral Vector Standard: Toward the Production Control and Standardization of Lentivirus-Based Gene Therapy Products.

Gene therapy is a rapidly evolving field. So far, there have been >2,400 gene therapy products in clinical trials and four products on the market. A prerequisite for producing gene therapy products is ensuring their quality and safety. This requires appropriately controlled and standardized production and testing procedures that result in consistent safety and efficacy. Assuring the quality and safety of lentivirus-based gene therapy products in particular presents a great challenge because they are cell-based multigene products that include viral and therapeutic proteins as well as modified cells. In addition to the continuous refinement of a product, changes in production sites and manufacturing processes have become more and more common, posing challenges to developers regarding reproducibility and comparability of results. This paper discusses the concept of developing a first World Health Organization International Standard, suitable for the standardization of assays and enabling comparison of cross-trial and cross-manufacturing results for this important vector platform. The standard will be expected to optimize the development of gene therapy medicinal products, which is especially important, given the usually orphan nature of the diseases to be treated, naturally hampering reproducibility and comparability of results.

Simultaneous Analysis of Secondary Structure and Light Scattering from Circular Dichroism Titrations: Application to Vectofusin-1.

Circular Dichroism data are often decomposed into their constituent spectra to quantify the secondary structure of peptides or proteins but the estimation of the secondary structure content fails when light scattering leads to spectral distortion. If peptide-induced liposome self-association occurs, subtracting control curves cannot correct for this. We show that if the cause of the light scattering is independent from the peptide structural changes, the CD spectra can be corrected using principal component analysis (PCA). The light scattering itself is analysed and found to be in good agreement with backscattering experiments. This method therefore allows to simultaneously follow structural changes related to peptide-liposome binding as well as peptide induced liposome self-association. We apply this method to study the structural changes and liposome binding of vectofusin-1, a transduction enhancing peptide used in lentivirus based gene therapy. Vectofusin-1 binds to POPC/POPS liposomes, causing a reversal of the negative liposome charge at high peptide concentrations. When the peptide charges exactly neutralise the lipid charges on both leaflets reversible liposome self-association occurs. These results are in good agreement with biological observations and provide further insight into the conditions required for efficent transduction enhancement.

Collagen epitope expression on B cells is sufficient to confer tolerance to collagen-induced arthritis.

BackgroundThe mechanisms underlying tolerance induction and maintenance in autoimmune arthritis remain elusive. In a mouse model of rheumatoid arthritis, collagen type II (CII)-induced arthritis, we explore the contribution of B cells to antigen-specific tolerance.MethodsTo generate expression of the CII-peptide specifically on B-cell major histocompatibility complex type II, lentiviral-based gene therapy including a B-cell-specific Igk promoter was used.ResultsPresentation of the CII-peptide on B cells significantly reduced the frequency and severity of arthritis as well as the serum levels of CII -specific IgG antibodies. Further, both frequency and suppressive function of regulatory T cells were increased in tolerized mice. Adoptive transfer of regulatory T cells from tolerized mice to naïve mice ameliorated the development of CII-induced arthritis.ConclusionOur data suggest that endogenous presentation of the CII-peptide on B cells is one of the key contributors to arthritis tolerance induction and maintenance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1037-7) contains supplementary material, which is available to authorized users.

Gene Therapy Induces Antigen-Specific Tolerance in Experimental Collagen-Induced Arthritis.

Here, we investigate induction of immunological tolerance by lentiviral based gene therapy in a mouse model of rheumatoid arthritis, collagen II-induced arthritis (CIA). Targeting the expression of the collagen type II (CII) to antigen presenting cells (APCs) induced antigen-specific tolerance, where only 5% of the mice developed arthritis as compared with 95% of the control mice. In the CII-tolerized mice, the proportion of Tregs as well as mRNA expression of SOCS1 (suppressors of cytokine signaling 1) increased at day 3 after CII immunization. Transfer of B cells or non-B cell APC, as well as T cells, from tolerized to naïve mice all mediated a certain degree of tolerance. Thus, sustainable tolerance is established very early during the course of arthritis and is mediated by both B and non-B cells as APCs. This novel approach for inducing tolerance to disease specific antigens can be used for studying tolerance mechanisms, not only in CIA but also in other autoimmune diseases.

709. Characterization of Nanoparticles in Lentiviral Vector Preparations

The potential of lentivirus-based gene therapy vectors for the treatment of severe genetic diseases using genetically modified CD34+ cells and hematological malignancies using chimeric antigen receptor T-cells (CAR-T) is supported by recent positive data in clinical trials showing promising therapeutic benefits and safety. The progress from early-to-late stage clinical development requires enhanced characterization of the purified lentivirus vector product. Lentiviral vector preparations are complex in nature and contain a heterogeneous mixture of transduction-competent as well as transduction-deficient virus particles. In addition, lentiviral vector production utilizes host cells that can produce not only the viral particles of interest, but a variety of closely-related impurities that include exosomes and microvesicles. These cell-derived impurities can overlap key biophysical and biochemical attributes of the lentiviral vector, including size, net charge and composition, making them challenging to analyze. We used a variety of analytical tools to further characterize lentiviral vector preparations in terms of size distribution, particle counts and to determine the total particle (viral or non-viral) to infectious particle ratio. These tools included Nanoparticle Tracking Analysis (Nanosight), Field-Flow Fractionation coupled to Multi-Angle Light Scattering (FFF-MALS), and p24-ELISA. Particle protein composition was evaluated by several standard orthogonal protein quantitation assays. Characterization of nanoparticle subpopulations is important as it can support the refinement of manufacturing processes. The impact of variation of these parameters on lentivirus vector performance still remains largely unknown, so interpretations of the results must be carefully assessed and further study is warranted.

Culture Of Mobilized Rhesus Macaque CD34+ Cells In Hypoxic Conditions Does Not Improve Lentiviral Transduction Efficiency In Long-Term Repopulating Hematopoietic Stem Cells

Low oxygen levels have been proposed to play a fundamental role in the maintenance of normal hematopoietic stem cells (HSC) function. We investigated whether culture of rhesus macaque mobilized CD34+ cells under low oxygen conditions (1-5% O2) could improve lentiviral transduction efficiency in HSCs compared to culture under atmospheric O2 conditions (21%). Mobilized CD34+ cells from 4 animals were prestimulated for 12 hours in the presence of cytokines and subsequently divided in two equal fractions for transduction with SIN-lentiviral vectors (MOI=100) expressing GFP or TdTomato fluorescent proteins for 48 hours. In 3 animals, cells were transduced with GFP vectors in hypoxia and TdTomato vectors in normoxia. These conditions were reversed in one animal. A portion of the transduced cells was used for phenotypic, cell cycle, clonogenic and apoptosis assays. The remaining cells from both fractions were combined and injected intravenously in lethally irradiated rhesus macaques and in vivo gene marking was measured in peripheral blood granulocytes for up to 21 months after transplantation. The numbers of total live cells and CD34+ cells after 2.5 days in culture were not significantly different compared to baseline for both hypoxic and normoxic conditions (p=0.85 and p=0.84, respectively). Similarly, numbers of CD34+CD38- cells were stable compared to baseline in hypoxia and normoxia (p=0.94). After 2.5 days of culture, the total number of colony-forming cells increased 1.4-fold under both hypoxic and normoxic conditions (p=0.69) compared to freshly isolated CD34+ cells. At baseline, the majority (50.6 + 4.4%) of the CD34+ cells were in the G0 phase of the cell cycle. After 2.5 days in culture under hypoxic or normoxic conditions, the percentages of cells in G0 were 19.9 + 8.8% and 18.6 + 5.9%, respectively (p=0.82). The differences in percentages of cells in the G1 and S/G2/M phases of the cell cycle were also not statistically significant (p=0.175 and p=0.732, respectively). The pO2 had no impact on cell death (18.2 + 5.3% in hypoxia and 16.8 + 4.8% in normoxia, p=0.69) or on the percentages of apoptotic cells (11.1 + 6.7% in hypoxia and 8.4 + 2.3% in normoxia, p=0.47). When considering the bulk of CD34+ cells after transduction with lentiviral vectors, the percentages of GFP-marked cells were consistently higher (range 1.8 to 2.2-fold, mean 2.1-fold) compared to TdTomato-marked cells independently of the transduction conditions used, consistent with a 2.1-fold intrinsic superiority of GFP-based lentiviral vectors compared to TdTomato vectors at equivalent MOI. Taking into account the inherent vector differences, transduction efficiencies were similar in normoxia (70.7 + 11.7%) and hypoxia (68.0 + 16.3%) (p=0.80). When adjusted for the intrinsic superiority of GFP-based vectors, the contribution to long-term in vivo gene marking measured by flow cytometry and quantitative PCR was comparable between cells transduced in 21% pO2 (9.0 + 4.5%) and 5% pO2 conditions (8.4 + 4.9%) (p=0.87). Consistent with these data, GFP and TdTomato-labeled hematopoietic cells were equally visualized, using a confocal/2-photon hybrid microscopy approach, in BM biopsy specimens collected at various time points after transplantation. Given recent evidence suggesting that lower oxygen concentrations (<1.5%) may be required to stabilize HIF-1a, a key sensor of hypoxic conditions, transduction was performed under extreme hypoxic conditions (1% O2) in one animal. While the overall viability of the bulk CD34+ cells after 2.5 days of transduction under extreme hypoxia was comparable to cells cultured in normoxia, their contribution to long-term in vivo marking was negligible (0.3%) compared to cells transduced under normoxic conditions (2.4%), suggesting toxicity of very low oxygen levels on HSCs. Overall, when current methodologies used for the genetic manipulation of HSCs for gene therapy applications were performed under hypoxic conditions, susceptibility to lentiviral transduction of CD34+ cells was not ameliorated compared to cells cultured in 21% O2 conditions in the rhesus macaque transplantation model. The short culture times (2-3 days) used in recent lentivirus-based gene therapy clinical trials and replicated in this study may not be sufficient to impact the phenotype of long-term repopulating HSCs and do not warrant incorporation of hypoxia in current gene therapy protocols. Disclosures:No relevant conflicts of interest to declare.

Characterization of Lentiviral Vector Production Using Microwell Suspension Cultures of HEK293T-Derived Producer Cells

ProSavin(®) is a lentiviral vector (LV)-based gene therapy for Parkinson's disease. ProSavin(®) is currently in a Phase I/II clinical trial using material that was generated by transient transfection of adherent human embryonic kidney (HEK)293T cells. For future large-scale productions of ProSavin(®), we have previously reported the development and characterization of two inducible producer cell lines, termed PS5.8 and PS46.2. PS46.2 has been successfully adapted to grow in suspension cultures. The present study describes the creation of a small-scale (<2 ml) microwell-based experimental platform for the parallel investigation of ProSavin(®) production using suspension-adapted PS46.2. This is combined with statistical design of experiments (DoE) techniques to enable rapid characterization of the process conditions that impact cell growth and LV production. The effects of postinduction period, microwell liquid fill volume, and concentration of inducer (doxycycline) on ProSavin(®) titer and the particle:infectivity (P:I) ratio was investigated using three rounds of DoE, in order to identify appropriate factor ranges and optimize production conditions. We identified an optimal "harvest window" between approximately 26-46 hr within which maximal titers of around 6×10(4) transducing units (TU)/ml were obtained (an approximately 30-fold improvement compared to starting microwell conditions), providing that the fill volume was maintained at or below 1 ml and the doxycycline concentration was at least 1.0 μg/ml. Insights from the microwell studies were subsequently used to rapidly establish operating conditions for ProSavin(®) production in a 0.5-L wave bioreactor culture. The information presented herein thus aids the design and evaluation of scalable production processes for LVs.

Antigen-Specific Gene Therapy after Immunisation Reduces the Severity of Collagen-Induced Arthritis

Reestablishment of tolerance induction in rheumatoid arthritis (RA) would be an optimal treatment with few, if any, side effects. However, to develop such a treatment further insights in the immunological mechanisms governing tolerance are needed. We have developed a model of antigen-specific tolerance in collagen type II (CII) induced arthritis (CIA) using lentivirus-based gene therapy. The immunodominant epitope of CII was inserted into a lentivirus vector to achieve expression on the MHC class II molecule and the lentiviral particles were subsequently intravenously injected at different time points during CIA. Injection of lentiviral particles in early phases of CIA, that is, at day 7 or day 26 after CII immunisation, partially prevented development of arthritis, decreased the serum levels of CII-specific IgG antibodies, and enhanced the suppressive function of CII-specific T regulatory cells. When lentiviral particles were injected during manifest arthritis, that is, at day 31 after CII immunisation, the severity of arthritis progression was ameliorated, the levels of CII-specific IgG antibodies decreased and the proportion of T regulatory cells increased. Thus, antigen-specific gene therapy is effective when administered throughout the inflammatory course of arthritis and offers a good model for investigation of the basic mechanisms during tolerance in CIA.

Stem cell-based approaches to treating HIV infection

Stem cell-based strategies for treating HIV-infected individuals represent a novel approach toward reconstituting the ravaged immune system with the ultimate aim of clearing the virus from the body. Genetic modification of human hematopoietic stem cells to produce cells that are either resistant to infection, cells that produce lower amounts of infectious virus, or cells that specifically target the immune response against the virus are currently the dominant strategies under development. This review focuses on the understanding of stem cell-based approaches that are under investigation and the rationale behind such approaches. Significant progress has recently been made utilizing stem cell-based approaches to treat HIV infection. Ideally, a successful strategy would result in immune clearance of the virus from the body as well long-term restoration of overall immune responses to successfully combat everyday environmental antigens. Two recent clinical trails illustrate how new advances in stem cell-based approaches may propel this field forward to clinical reality: one that demonstrates that large-scale gene therapy trials can be performed in a conventional, reproducible manner; and one that demonstrates the utilization of a multipronged approach using lentiviral-based gene therapy vectors. These clinical trails serve as the foundation for the development of other technologies, discussed here, that are currently in preclinical development. Recent advances using stem cell-based approaches to treat HIV infection have provided the impetus for a renewed and expanded interest in the development of new cell-based strategies to treat HIV infection as well as a variety of other diseases.

Lentiviral Vectors That Express UGT1A1 in Liver and Contain miR-142 Target Sequences Normalize Hyperbilirubinemia in Gunn Rats

Crigler-Najjar type 1 (CN-I) is an inherited liver disease caused by an absence of bilirubin-uridine 5'-diphosphate-glucuronosyltransferase (UGT1A1) activity. It results in life-threatening levels of unconjugated bilirubin, and therapeutic options are limited. We used adult Gunn rats (an animal model of the disease) to evaluate the efficiency of lentiviral-based gene therapy to express UGT1A1 in liver. Gunn rats were given intraportal injections of VSVG-pseudotyped lentiviral vectors that encode UGT1A1 under the control of a liver-specific transthyretin promoter (mTTR.hUGT1A1); this vector does not contain target sequences for miR-142, a microRNA that is expressed specifically in hematopoietic cells. Rats were also injected with the vector mTTR.hUGT1A1.142T, which contains 4 copies of the miR-142 target sequences; its messenger RNA should be degraded in antigen-presenting cells. Bilirubinemia was monitored, and the presence of transduced hepatocytes was analyzed by quantitative polymerase chain reaction. Vector expression was tested in vitro in rat hematopoietic cells. In Gunn rats, bilirubin levels normalized 2 weeks after administration of mTTR.hUGT1A1. However, hyperbilirubinemia resumed 8 weeks after vector administration, concomitant with the induction of an immune response. In contrast, in rats injected with mTTR-UGT1A1.142T, bilirubin levels normalized for up to 6 months and transduced cells were not eliminated. Lentiviral vectors that express UGT1A1 reduce hyperbilirubinemia in immunocompetent Gunn rats for at least 6 months. The immune response against virally expressed UGT1A1 can be circumvented by inclusion of miR-142 target sequences, which reduce vector expression in antigen-presenting cells. This lentiviral-based gene therapy approach might be developed to treat patients with CN-I.