Gene Therapy In Models Research Articles

CRISPR/Cas9-mediated somatic and germline gene correction to restore hemostasis in hemophilia B mice.

Hemophilia B (HB) is an X-linked disorder caused by defects of F9 encoded coagulation factor IX, which is an ideal model for gene therapy. Most existing HB gene therapies are based on viral mediated gene supplementation, which could increase immunoreaction. In this study, CRISPR/Cas9 system was used for gene correction in an F9 mutant HB mouse model in both adult mice (in vivo) and in germline cells (ex vivo). In vivo, naked Cas9-sgRNA plasmid and donor DNA were delivered to HB mice livers to recover the mutation via hydrodynamic tail vein (HTV) injection. 62.5% of the HTV-treated mice showed a detectable gene correction (>1%) in the F9 alleles of hepatocytes, which was sufficient to remit the coagulation deficiency. Ex vivo, three different forms of Cas9 were microinjected into germline cells of HB mice to investigate their efficiency and safety in gene correction. Cas9 protein showed higher gene recovery rates, less embryo toxicity, and lower mosaic repair percentage, making it more suitable for germline gene therapy. Our study strongly supports that CRISPR/Cas9-mediated genome editing is feasible in gene therapy of genetic disorders.

Overexpression of Thioredoxin1 enhances functional recovery in a mouse model of hind limb ischemia

BackgroundThere is keen interest in finding nonsurgical treatments for peripheral vascular disease (PVD). Previously, we demonstrated that selective activation of Thioredoxin1 (Trx1), a 12-kDa cytosolic protein, initiates redox-dependent signaling and promotes neovascularization after ischemic heart disease. Therefore, Trx1 might possess immense potential to not only treat murine hind limb ischemia (HLI) through effective angiogenesis but also provide PVD patients with nonsurgical therapy to enhance neovascularization and improve blood perfusion. MethodsTo determine whether activation of Trx1 increases blood perfusion in HLI, two different strategies were used—gene therapy and transgenic model system. In adenoviral-mediated gene therapy, 8- to 12-wk-old mice were divided into two groups: (1) control Adeno-LacZ (Ad-LacZ) and (2) Adeno-Thiroedoxin1 (Ad-Trx1). The mice underwent surgical intervention to induce right HLI followed by injection with Ad-LacZ or Ad-Trx1, respectively. In the second strategy, we used wild-type and transgenic mice overexpressing Trx1 (Trx1Tg/+). All the animals underwent Doppler imaging for the assessment of limb perfusion followed by immunohistochemistry and Western blot analysis. ResultsSignificant increases in perfusion ratio were observed in all the Trx1 overexpressed groups compared with their corresponding controls. Expressions of heme oxygenase-1, vascular endothelial growth factor, and the vascular endothelial growth factor receptors Flk-1 and Flt-1 were increased in Trx1 overexpressed mice compared with their respective controls. Blood perfusion in the ischemic limb gradually improved and significantly recovered in Trx1Tg/+ and Ad-Trx1 groups compared with their corresponding controls. The capillary and arteriolar density in the ischemic zone were found to be higher in Trx1Tg/+ group compared with wild type. ConclusionsThe overall outcomes of our study demonstrate that Trx1 enhances blood perfusion and increases angiogenic protein expression in a rodent HLI model. These results suggest that Trx1 is a potential target for clinical trials and drug therapy for the treatment of PVD.

331 - In vitro differentiation of red blood cells from induced pluripotent stem cells as alternative resource for transfusion and a model for gene therapy

331 - In vitro differentiation of red blood cells from induced pluripotent stem cells as alternative resource for transfusion and a model for gene therapy

A murine model of suicide gene therapy in the brainstem demonstrates efficacy without toxic inflammation, suggesting possible use as a treatment for diffuse intrinsic pontine glioma (DIPG)

Abstract Diffuse intrinsic pontine glioma (DIPG) is a rare pediatric brainstem tumor that is the leading cause of death among pediatric brain malignancies. Despite over 200 clinical trials, no significant progress has been made, leading our lab to consider suicide gene therapy. The brainstem has particularly sensitive anatomy, creating the potential for toxic inflammation. Furthermore, the clinical standard of care for DIPG includes conventional radiation therapy (RT) followed by dexamethasone (DEX), each of which can alter the efficacy of gene therapy. In this study we found that a variety of gliomas, including human DIPG and murine glioma cell lines, are infectable in vitro with a replication-competent Moloney murine leukemia virus (MoMLV) (currently in Phase 2/3 clinical trials) and susceptible to ganciclovir (GCV)-mediated killing via the HSV thymidine kinase (tk) suicide gene. A B16 melanoma cell line expressing HSV tk implanted into the brainstem of mice, extended median survival with GCV therapy from 13 days to 38.5 days (p=.0006) with no signs of toxic inflammation. Pre-treatment with RT and DEX did not affect therapeutic efficacy. We have shown that GCV/TK therapy is mediated by NK and CD8 cells in subcutaneous tumors, and we are investigating whether a similar immunologic mechanism is involved in this brainstem model. These preclinical data will pave the way for novel clinical trials for the treatment of DIPG.

EIF3i activity is critical for endothelial cells in tumor induced angiogenesis through regulating VEGFR and ERK translation.

Translational control is a critical step in the regulation of gene expression. Accumulating evidence shows that translational control of a subgroup of mRNAs tends to be selective. However, our understanding of the function of selective translational control in endothelial cells is still incomplete. We found that a key translational regulator, eIF3i, is highly expressed in endothelial cells during embryonic and tumor angiogenesis. Knockdown of eIF3i restrained cell proliferation and migration in endothelial cells. In zebrafish angiogenesis model, eIF3i mutant endothelial cells could not respond to induction signals from tumor mass. Mechanistically, we showed that eIF3i knockdown reduced VEGFR/ERK signaling by down-regulating VEGFR2 and ERK protein expression. Gene therapy model suggested that the growth and metastasis of cancer cells were suppressed by eIF3i shRNA. Therefore, our work established a selective translational regulatory mechanism during tumor induced angiogenesis and suggested that targeting eIF3i may be applicable for anticancer therapy.

Fah Knockout Animals as Models for Therapeutic Liver Repopulation.

Several animal models of Fah deficiency have been developed, including mice, pigs and most recently rats. Initially, the murine models were developed with the intent to mirror the human disease for pathophysiologic and therapeutic studies. However, it soon became apparent that Fah-positive hepatocytes have a potent selective growth advantage in mutant liver and can extensively repopulate the diseased organ. For this reason, Fah mutant mice have become a workhorse for liver biology and are widely used in liver stem cell and hepatic gene therapy research. Immune deficient Fah-knockout mice can be repopulated with human hepatocytes, creating "mice with human livers". These chimeric animals have become an important preclinical model for infectious diseases, metabolism and gene therapy. The potent expansion of human hepatocytes in Fah knockout mice has given rise to the concept of using Fah mutants as living bioreactors to produce large quantities of fully mature hepatocytes. As a consequence, larger animal models of Fah deficiency have recently been developed.

HSC Clonal Dynamics after T-Cell Depletion in a Nonhuman Primate Model of Lentiviral Gene Therapy

Background:Hematopoietic stem cell (HSC) gene therapy is a new treatment paradigm that can potentially provide lifelong protection against HIV-1 infection. The basic principle is to genetically modify a patient's own HSC such that the progeny, including CD4+T cells and macrophages, are resistant to HIV-1. Given the expected low levels of the anti-HIV gene-marked cells in clinical studies, it is critical to understand how the vast number of HSC, each bearing a unique phenotype, and their mature progeny contribute to maintaining homeostatic regulation in HIV gene therapy settings. We have recently published novel and detailed insights about the long-term behavior patterns of individual HSC followed for 4 to 12 years post-transplant in our nonhuman primate (NHP) model of lentiviral gene therapy, revealing for the first time in primates the precise time point of HSC repopulation and the functional heterogeneity of HSCs (Kim, Cell Stem Cell, 2014; Goyal, BMC Biology, 2015). Consistent clonal behavior patterns have been observed in human gene therapy (Biasco, Cell Stem Cell, 2016), demonstrating the clinical relevance of our data. Here, in order to develop a systems-level understanding of HSC clonal dynamics in anti-HIV therapy settings requiring efficient immune recovery from T lymphopenia, we further analyzed HSC clonal repopulation after T-cell depletion in one of our NHP animals. Our study generated systems-level datasets useful for understanding the parameters of T-cell repopulation and homeostatic regulation in anti-HIV therapy, as well as other therapies requiring efficient immune recovery from disease- or treatment-induced T lymphopenia.Subjects and Methods:The HSC clonal behaviors in animal 95E132 have been well characterized for 16 years post-transplant. At the 16-year time point, this animal was treated with 8 doses (25 ug/kg/dose) of an anti-CD3e immunotoxin over 4 days. Hematopoietic recovery after T-cell depletion was monitored by a complete blood count, multi-color flow cytometry analysis, TCRv_ spectratyping, and a high-throughput lentiviral-tagging assay (Kim, Journal of Virology, 2010). HSC subtypes and their clonal behaviors were determined based on the clonal profiling of blood lineages, including CD4, CD8, CD20, CD14, and CD18 cells, over time.Results:The peripheral CD3+ T cells recovered in 2-3 months (Fig. 1B). Cytotoxic (CD8+) T-cell recovery was faster than that of T-helper cells. Effector and memory T cells expanded promptly after T-cell depletion, whereas the na•ve T cells had not recovered more than one year after CD3e-immunotoxin treatment. Clonal profiling analysis revealed a few dominant clones in the recovered T-cell compartment, while showing no notable clonal fluctuation in the CD18+ granulocytes over time (Fig. 1C-D). TCRv_ spectratyping showed a skewed T-cell receptor repertoire even a year after immunotoxin treatment.Conclusion:Our data can bolster our understanding of hematopoietic regulation in patients recovering from T lymphopenia. The data showed skewed a T-cell receptor repertoire and clonal dominance in the recovered T-cells after immunotoxin treatment in an aged animal, suggesting that T-cell recovery had occurred primarily as a result of peripheral T-cell expansion. A systems-level, clonal dynamics study of this extreme form of homeostatic regulation provides unique opportunities to identify and characterize the regenerative pathways, as well as the obstacles, that emerge in the process of restoring homeostasis after disease- or treatment-induced T-cell depletion. [Display omitted] DisclosuresDunbar:GSK/Novartis: Research Funding. Chen:Calimmune: Membership on an entity's Board of Directors or advisory committees.

Preclinical Development of Gene Therapy for X-Linked Severe Combined Immunodeficiency (SCID-X1)

X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the gene encoding the interleukin-2 receptor γ chain (IL2RG), and is characterized by lack of response to common γ-chain-dependent cytokines and profound defects in T-, B- and NK-cell functions. Previous gene therapy clinical trials based on autologous transplantation of hematopoietic stem/progenitor cells (HSPCs) genetically corrected with MLV-derived retroviral vectors expressing IL2RG in non-conditioned patients showed long-lasting restoration of T-cell immunity in most cases but resulted in vector-induced leukemia through enhancer-mediated insertional mutagenesis in 25% of patients. The use of an enhancer-less MLV vector to deliver the IL2RG gene caused no adverse events while retaining a significant clinical benefit. To increase the efficacy of gene therapy and reduce at the same time the probability of genotoxic insertional events, we developed a third-generation lentiviral vector carrying a codon-optimized human IL2RG cDNA under the control of the human EF1α-S promoter. The vector was VSV-G-pseudotyped and produced by transient transfection of 293T cells, followed by ion-exchange chromatography, tangential-flow filtration and formulation in HSC growth medium. Replacement of the native IL2RG open reading frame by a codon-optimized sequence resulted in a 3-fold increase in mRNA expression and a 1.5-fold increase in IL2RG protein expression per integrated vector copy. The performance of the vector was demonstrated in vitro by the restoration of a normal level of IL2RG mRNA or protein in a human IL2RG-deficient T-cell line at a VCN of 1 to 3, and by high efficiency (>80%) transduction of human mobilized CD34+ HSPCs and transgene expression with no impact on viability or clonogenic capacity.An in vitro immortalization assay (IVIM) showed a safe genotoxic profile, while the in vivo safety and efficacy of the vector was tested it in a preclinical model of SCID-X1 gene therapy based on transplantation of genetically corrected Lin- cells from IL2rg-/- donor mice into lethally-irradiated IL2rg-/--Rag2-/- recipients. The study showed biodistribution of the transgene in hematopoietic organs only, restoration of T, B and NK cell counts in bone marrow and peripheral blood, normalization of lymphoid organs (thymus and spleen) and a low frequency of hematopoietic malignancies, comparable to that of untreated animals, six months after transplantation.An extensive insertion site analysis was carried out in bone marrow, thymus and peripheral blood of individual or groups of animals to detect the presence of any clonal abnormality or skewing, and any deviation from a normal lentiviral integration pattern. The study showed the expected genomic integration profile and no signs of clonal dominance of transduced cells. Interestingly, analysis of >100,000 integration sites in pre- and post-transplant murine cells showed that lentiviral vectors target at high frequency a substantially different set of genes compared to human CD34+ cells, uncovering the need for appropriate controls and at the same time the limits in the predictive power of mouse-based genotoxicity studies, particularly those addressing proto-oncogene activation and clonal dominance.These studies will enable a multicenter phase-I/II clinical trial aimed at establishing the safety and clinical efficacy of lentiviral vector-mediated gene therapy for SCID-X1. The clinical protocol is based on transplantation of autologous CD34+ HSPCs transduced with the EF1a-IL2RGco lentiviral vector in infants after non-myeloablative conditioning, and aims at stable and sustained restoration of both T- and B-cell immunity. DisclosuresWilliams:Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees. Gaspar:Orchard therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mavilio:Adverum Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Consultancy, Research Funding.

Identification and Characterization of a Distinct, Evolutionarily Conserved HSC Phenotype Associated with and Predicting Multi-Lineage Engraftment

Current autologous stem cell transplantation and gene therapy strategies are limited by the inability to identify a true hematopoietic stem cell (HSC) propulation that reliably predicts engraftment. The ability to enrich and target a specific HSC pool and predict engraftment levels of the gene-modified cells would be a major advance, reducing manufacturing costs and off-target effects. Here we describe a distinct HSC phenotype, conserved between humans and nonhuman primates (NHP) which overcomes this limitation.We used our NHP stem cell transplantation and gene therapy model to study the engraftment potential of phenotypically distinct hematopoietic stem and progenitor cell (HSPC) subpopulations. We were able to identify an exclusive HSPC subpopulation capable of multi-lineage engraftment (Figure 1A and 1B). This HSPC subpopulation (denoted "I") accounts for ~3-5% of the entire CD34+ cell population in primed bone marrow, reducing the number of cells targeted for cell and gene therapy approaches by 20 to 30-fold. For autologous transplants, only 300-400K HSPCs/kg body weight were required to achieve rapid neutrophil and plateletet recovery within 9-10 and 19-20 days, respectively. Stable 30-35% gene-marking was obeserved in all blood lineages including T cell, B cell, NK cells, granulocyte, monocytes/macrophages, erythrocytes and platelets. Complete reconstitution of the bone marrow compartment was achieved within <100 days, with 31-33% gene-marking in HSCs as well as downstream progenitors from all lineages.Most importantly, and in support of the above described engraftment studies, the use of this HSPC population I phenotype allowed us to establish a reliable flow cytometry based analysis to assess and predict engraftment after HSC transplantion. This method predicted the onset of neutrophil and platelet recovery (Figure 1C and 1D). This assay was also able to predict the onset of NHP hematopoietic recovery for transplanted HSCs from different stem cell sources including steady state (unprimed) bone marrow, as well as gene-modified/transduced and ex vivo expanded HSCs. From this data, we were able to define the minimum cell number of HSCs/kg body weight required for sustained multi-lineage long-term engraftment with recovery of all blood cell lineages in the NHP transplant setting as 110,000 cells/kg. Importantly, we demonstrated that this cell population and phenotype is conserved between NHP and human hematopoiesis (Radtke et al. 2016, submitted).In summary, we identified a distinct and evolutionarily conserved HSC phenotype that is associated with multi-lineage engraftment in nonhuman primates and also reliably predicted the engraftment kinetics after transplant. This will greatly facilitate evaluation and "potency" of autolgous stem cell products especially after stem cell expansion or gene therapy. In addition, it will allow for an improved enrichment of the target cells for gene therapy and gene editing protocols and thus likely improve efficacy and safety. [Display omitted] DisclosuresAdair:Rocket Pharmaceuticals: Consultancy, Equity Ownership. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Research Funding.

Autoregulated expression of p53 from an adenoviral vector confers superior tumor inhibition in a model of prostate carcinoma gene therapy

ABSTRACTAlternative treatments for cancer using gene therapy approaches have shown promising results and some have even reached the marketplace. Even so, additional improvements are needed, such as employing a strategically chosen promoter to drive expression of the transgene in the target cell. Previously, we described viral vectors where high-level transgene expression was achieved using a p53-responsive promoter. Here we present an adenoviral vector (AdPGp53) where p53 is employed to regulate its own expression and which outperforms a traditional vector when tested in a model of gene therapy for prostate cancer. The functionality of AdPGp53 and AdCMVp53 were compared in human prostate carcinoma cell lines. AdPGp53 conferred greatly enhanced levels of p53 protein and induction of the p53 target gene, p21, as well as superior cell killing by a mechanism consistent with apoptosis. DU145 cells were susceptible to induction of death with AdPGp53, yet PC3 cells were quite resistant. Though AdCMVp53 was shown to be reliable, extremely high-level expression of p53 offered by AdPGp53 was necessary for tumor suppressor activity in PC3 and DU145. In situ gene therapy experiments revealed tumor inhibition and increased overall survival in response to AdPGp53, but not AdCMVp53. Upon histologic examination, only AdPGp53 treatment was correlated with the detection of both p53 and TUNEL-positive cells. This study points to the importance of improved vector performance for gene therapy of prostate cancer.

BET bromodomain inhibition enhances T cell persistence and function in adoptive immunotherapy models.

Adoptive immunotherapy is a potentially curative therapeutic approach for patients with advanced cancer. However, the in vitro expansion of antitumor T cells prior to infusion inevitably incurs differentiation towards effector T cells and impairs persistence following adoptive transfer. Epigenetic profiles regulate gene expression of key transcription factors over the course of immune cell differentiation, proliferation, and function. Using comprehensive screening of chemical probes with defined epigenetic targets, we found that JQ1, an inhibitor of bromodomain and extra-terminal motif (BET) proteins, maintained CD8+ T cells with functional properties of stem cell-like and central memory T cells. Mechanistically, the BET protein BRD4 directly regulated expression of the transcription factor BATF in CD8+ T cells, which was associated with differentiation of T cells into an effector memory phenotype. JQ1-treated T cells showed enhanced persistence and antitumor effects in murine T cell receptor and chimeric antigen receptor gene therapy models. Furthermore, we found that histone acetyltransferase p300 supported the recruitment of BRD4 to the BATF promoter region, and p300 inhibition similarly augmented antitumor effects of the adoptively transferred T cells. These results demonstrate that targeting the BRD4-p300 signaling cascade supports the generation of superior antitumor T cell grafts for adoptive immunotherapy.

A gene therapy model to treat liver-implanted tumors.

An expression vector based on the Semliki Forest Virus (SFV), an alpha virus replicating in the cytoplasm of infected cells originally discovered in the Semliki Forest in Uganda, was developed to express interleukin 12 (IL-12) and SVF-IL-12 were shown to induce potent antitumor response against liver implanted colon tumor cells in immunocompetent mice in a recent study by Jose I. Quetglas and co-investigators [J. I. Quetglas, et. al, J. of Immunology 2013]. The researchers reported that the tumor-specific CD 8+ T cells in responder mice persisted at greater numbers and for a long period of time while maintaining enhanced effector functions in contrast to those of non-responder animals. The long-lasting persistence and activity of tumor-specific CD8+ T cells from responder mice might be mediated by the production of IFN-alpha by cells infected by SFV as the replication of this virus can induce type I IFN response. We proposed a mathematical model based on a set of ordinary differential equations with an attempt to describe the SFV-IL-12 antitumor efficacy. The project was supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and the West Virginia Higher Education Policy Commission Division of Science and Research SURE Grant Program.

284. Long-Term Therapeutic Immune Reconstitution in XSCID Canine Model via In Vivo Foamy Virus Delivery of Common Gamma Chain

X-linked combined immunodeficiency disease (XSCID) is caused by mutation in the common gamma chain, γC (interleukin-2 receptor subunit gamma, IL2RG) in both humans and canines. It is characterized by the inability of T-cell development leading to absence of T-cells in peripheral blood, lack of T-cell mediated immune response, low IgA and IgG levels, and early infant mortality. In the 1990s, human XSCID clinical trials utilizing gamma-retroviral vectors to deliver the IL2RG gene caused leukemia in 5 out of 20 patients due to vector integration in or near proto-oncogenes. Recent studies showed Foamy virus based vectors as an excellent alternative for in vivo gene-therapy because it is non-pathogenic in humans while exhibiting increased serum stability and favorable integration pattern. Previously, we have demonstrated CD3+ T-cell reconstitution in the canine model via intravenous injection of foamy virus expressing human elongation factor-1 alpha promoter (Ef1α)-yC. Unfortunately, the treated animals contained a low number of gene corrected progenitors at a sub-therapeutic level. Here, we achieved long-term therapeutic immune-reconstitution by intravenous delivery of a human phosphoglycerate kinase promoter (Pgk)-mediated γC foamy viral vector into XSCID neonatal canines. Long-term (2 years) post-injection follow-up demonstrated therapeutic levels of CD3+ T-cell expansion. Within the T-cell population, gene correction with Pgk-γC stabilized at ~80%. We validated T-cell functionality by using spectratyping analysis, which exhibited a diverse repertoire of receptor gene rearrangement. Retroviral integration site analysis (RIS) indicated polyclonal contribution to the reconstituted T-cells. Immunoglobulin ELISA assays showed that IgA and IgG levels in peripheral blood are comparable to normal healthy controls. We immunized the gene-corrected canine recipients with bacteriophage ϕx174 and confirmed production of specific IgG antibodies, showing the ability for isotype switching in B-lymphocytes. Currently, the gene-corrected canines exhibit comparable health and physical attributes to normal controls. Furthermore, semen from the gene-corrected male canine was used via artificial insemination to produce a litter of viable offsprings. In summary, our data demonstrate that Pgk-γC foamy viral vector delivered long-term therapeutic gene correction in a large-animal model for XSCID gene therapy. Most importantly, these results indicate that in vivo Pgk-γC foamy vector administration is a viable option for long-term immune reconstitution in future XSCID human clinical trials.

607. Organotypic Culture of Adult Porcine Retina as an In Vitro Screening Model for AAV-Mediated Gene Therapy in Ophthalmology

Introduction: Gene therapy studies in vivo using animal models provide critical information regarding vector specificity, therapeutic efficacy, and safety. However, long durations to observe transgene expression, high degree of variability between animals, and reduced control of experimental conditions often leads to large sample sizes and expense. Organotypic cultures of the retina can provide an efficient in vitro model with a high degree of throughput and control that can be used for pharmacological screening. These explants preserve complex intercellular processes and communications among the neural retinal layers with minimal manipulation of tissue architecture and molecular pathways, making it a useful model in target-tissue validation of adeno-associated virus (AAV) vector variants. Porcine retina has similar anatomical and physiologic features to the human retina and is therefore a suitable surrogate for preclinical testing in ophthalmology. Methods: We have developed a porcine explant culture system from retinas isolated from adult pigs as a model to screen novel rAAV vectors for transduction efficiency and cellular tropism. Pig eyes were enucleated and the neural retina dissected away from the pigment epithelium within 1-2 hours postmortem. Tissue was cut into 4 mm pieces and placed photoreceptor layer down on polycarbonate transwells, and cultured in Neurobasal-A medium supplemented with B-27. Viability of the explant cultures was examined over time by gross morphology, quantification of glucose-6-phosphate dehydrogenase release from dying cells, and TUNEL assay. Transductions with AAV vectors were performed on retinal explants on the day the cultures were started, and transgene expression was analyzed by live cell imaging and immunofluorescence (IF) staining, or ELISA from tissue supernatants. Results: Retinal explants were viable for up to 4 weeks and IF staining of retinal protein markers including β-III-tubulin, Chx10, glutamine synthetase, rhodopsin and L/M opsin suggested that most retinal cell types were preserved in vitro, with tissue architecture closely resembling that of intact in vivo retina. Transduction with different rAAV serotypes encoding GFP displayed protein expression at 3 days, with gradually rising levels during the first 2 weeks following transduction. Furthermore, IF staining using retinal cell markers revealed differential cellular transgene expression from the AAV vectors encoding various cell-specific or ubiquitous promoters under investigation. Finally, high levels of secreted soluble VEGFR-1 protein expressed from rAAV were detected from explant culture media samples with expression increasing over a period of 3 weeks. Conclusions: Our results demonstrate viability of retinal explants for up to 4 weeks and efficient rAAV transduction with various serotypes. Our organotypic porcine retinal explant model facilitates the evaluation of efficacy, cellular tropism, and promoter selectivity of rAAV vectors in a fast, reproducible, and economical manner, contributing to a reduction in the use of animals for the development of therapies in ophthalmology.

Inducible scAAV2.GRE.MMP1 lowers IOP long-term in a large animal model for steroid-induced glaucoma gene therapy.

Current treatment of glaucoma relies on administration of daily drops or eye surgery. A gene therapy approach to treat steroid-induced glaucoma would bring a resolution to millions of people worldwide that depend on glucocorticoid therapy for a myriad of inflammatory disorders. Previously, we had characterized a short-term Adh.GRE.MMP1 gene vector for the production of steroid-induced MMP1 in the trabecular meshwork and tested reduction of elevated intraocular pressure (IOP) in a sheep model. Here we conducted a trial transferring the same transgene cassette to a clinically safe vector (scAAV2), and extended the therapeutic outcome to longer periods of times. No evidence of ocular and/or systemic toxicity was observed. Viral genome distributions showed potential re-inducible vector DNAs in the trabecular meshwork (0.4 vg/cell) and negligible copies in six major internal organs (0.00002-0.005 vg/cell). Histological sections confirmed successful transduction of scAAV2.GFP to the trabecular meshwork. Optimization of the sheep steroid–induced hypertensive model revealed that topical ophthalmic drug difluprednate 0.05% (durezol) induced the highest IOP elevation in the shortest time. This is the first efficacy/toxicity study of a feasible gene therapy treatment of steroid-induced hypertension using clinically accepted scAAV vectors in a large animal model.

Total body irradiation must be delivered at high dose for efficient engraftment and tolerance in a rhesus stem cell gene therapy model

Reduced intensity conditioning (RIC) is desirable for hematopoietic stem cell (HSC) gene therapy applications. However, low gene marking was previously observed in gene therapy trials, suggesting that RIC might be insufficient for (i) opening niches for efficient engraftment and/or (ii) inducing immunological tolerance for transgene-encoded proteins. Therefore, we evaluated both engraftment and tolerance for gene-modified cells using our rhesus HSC gene therapy model following RIC. We investigated a dose de-escalation of total body irradiation (TBI) from our standard dose of 10Gy (10, 8, 6, and 4Gy), in which rhesus CD34+ cells were transduced with a VSVG-pseudotyped chimeric HIV-1 vector encoding enhanced green fluorescent protein (GFP) (or enhanced yellow fluorescent protein (YFP)). At ~6 months after transplantation, higher-dose TBI resulted in higher gene marking with logarithmic regression in peripheral blood cells. We then evaluated immunological tolerance for gene-modified cells, and found that lower-dose TBI allowed vigorous anti-GFP antibody production with logarithmic regression, while no significant anti-VSVG antibody formation was observed among all TBI groups. These data suggest that higher-dose TBI improves both engraftment and immunological tolerance for gene-modified cells. Additional immunosuppression might be required in RIC to induce tolerance for transgene products. Our findings should be valuable for developing conditioning regimens for HSC gene therapy applications.

Inducible scAAV2.GRE.MMP1 lowers IOP long-term in a large animal model for steroid-induced glaucoma gene therapy

Inducible scAAV2.GRE.MMP1 lowers IOP long-term in a large animal model for steroid-induced glaucoma gene therapy

Pax3-induced expansion enables the genetic correction of dystrophic satellite cells.

BackgroundSatellite cells (SCs) are indispensable for muscle regeneration and repair; however, due to low frequency in primary muscle and loss of engraftment potential after ex vivo expansion, their use in cell therapy is currently unfeasible. To date, an alternative to this limitation has been the transplantation of SC-derived myogenic progenitor cells (MPCs), although these do not hold the same attractive properties of stem cells, such as self-renewal and long-term regenerative potential.MethodsWe develop a method to expand wild-type and dystrophic fresh isolated satellite cells using transient expression of Pax3. This approach can be combined with genetic correction of dystrophic satellite cells and utilized to promote muscle regeneration when transplanted into dystrophic mice.ResultsHere, we show that SCs from wild-type and dystrophic mice can be expanded in culture through transient expression of Pax3, and these expanded activated SCs can regenerate the muscle. We test this approach in a gene therapy model by correcting dystrophic SCs from a mouse lacking dystrophin using a Sleeping Beauty transposon carrying the human μDYSTROPHIN gene. Transplantation of these expanded corrected cells into immune-deficient, dystrophin-deficient mice generated large numbers of dystrophin-expressing myofibers and improved contractile strength. Importantly, in vitro expanded SCs engrafted the SC compartment and could regenerate muscle after secondary injury.ConclusionThese results demonstrate that Pax3 is able to promote the ex vivo expansion of SCs while maintaining their stem cell regenerative properties.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0061-7) contains supplementary material, which is available to authorized users.

Synergy between rapamycin and FLT3 ligand enhances plasmacytoid dendritic cell–dependent induction of CD4+CD25+FoxP3+ Treg

AbstractCD4+CD25+FoxP3+ regulatory T cells (Treg) are critical elements for maintaining immune tolerance, for instance to exogenous antigens that are introduced during therapeutic interventions such as cell/organ transplant or gene/protein replacement therapy. Coadministration of antigen with rapamycin simultaneously promotes deletion of conventional CD4+ T cells and induction of Treg. Here, we report that the cytokine FMS-like receptor tyrosine kinase ligand (Flt3L) enhances the in vivo effect of rapamycin. This occurs via selective expansion of plasmacytoid dendritic cells (pDCs), which further augments the number of Treg. Whereas in conventional DCs, rapamycin effectively blocks mammalian target of rapamycin (mTOR) 1 signaling induced by Flt3L, increased mTOR1 activity renders pDCs more resistant to inhibition by rapamycin. Consequently, Flt3L and rapamycin synergistically promote induction of antigen-specific Treg via selective expansion of pDCs. This concept is supported by the finding that Treg induction is abrogated upon pDC depletion. The combination with pDCs and rapamycin is requisite for Flt3L/antigen-induced Treg induction because Flt3L/antigen by itself fails to induce Treg. As coadministering Flt3L, rapamycin, and antigen blocked CD8+ T-cell and antibody responses in models of gene and protein therapy, we conclude that the differential effect of rapamycin on DC subsets can be exploited for improved tolerance induction.

294. Myeloablative Conditioning Is Required for Efficient Engraftment of Gene-Modified Cells and Prevention of Antibody Production Against Transgene Products in a Rhesus Stem Cell Gene Therapy Model

Reduced intensity conditioning (RIC) regimens are desirable for hematopoietic stem cell (HSC)-targeted gene therapy. However, in previous gene therapy trials, low gene marking was reported in peripheral blood cells, raising the question that RIC might be insufficient for (1) opening niches for efficient engraftment and (2) inducing immunological tolerance to transgenes. Therefore, we sought to evaluate both engraftment and tolerance for genetically modified cells using our rhesus HSC gene therapy model following RIC.We performed a dose de-escalation of total body irradiation (TBI) (10, 8, 6, and 4Gy) as RIC transplantation (total 19 animals), in which mobilized rhesus CD34+ cells were transduced with a VSV-G-pseudotyped chimeric HIV-1 vector encoding GFP (or YFP) and these cells were transplanted into autologous animals following TBI. We evaluated GFP-positive rates (%GFP), average vector copy number per cell (VCN), and antibody titers against GFP and VSV-G in peripheral blood 6 months after transplantation.When we evaluated in vitro %GFP in transduced rhesus CD34+ cells, efficient transduction (22-71%) was observed among all TBI groups (without correlation). After transplantation of the transduced CD34+ cells, GFP-positive cells were detected in peripheral blood cells in all animals. Increasing doses of TBI resulted in higher %GFP with logarithmic regression in both granulocytes (R2=0.64, p<0.01) and lymphocytes (R2=0.67, p<0.01)(Figure). Similar results were observed between TBI dose and VCNs in both granulocytes (R2=0.54, p<0.01) and lymphocytes (R2=0.52, p<0.01). These data suggest that higher doses of TBI improve engraftment of gene-modified hematopoietic repopulating cells.To evaluate immunological tolerance for gene-modified cells, we measured both anti-GFP and anti-VSV-G antibody titers using serum samples from transplanted animals. Lower dose TBI allowed anti-GFP antibody production with logarithmic regression (R2=0.84, p<0.01)(Figure), while no significant effect of anti-VSV-G antibody was observed among all TBI groups (R2=0.003, p=0.87). These data demonstrate that higher dose TBI is important to induce immunological tolerance for gene-modified cells in a rhesus transplantation model.In summary, higher dose of TBI increased gene marking levels in transplanted rhesus macaques, while lower dose of TBI resulted in anti-GFP antibody production. Additional immunosuppressive therapy might be required in RIC to induce immunological tolerance for transgene products. Our findings should be valuable to consider conditioning regimens for clinical gene therapy. View Large Image | Download PowerPoint Slide