ALBERT

Description: Using gene therapy to protect hematopoietic stem cells (HSC) from alkylating agents used in the treatment of malignant disease is an attractive strategy to alleviate prolonged neutropenia and thrombocytopenia that is dose-limiting. Both adult and pediatric patients with glioblastomas urgently need improved therapeutic strategies since even with aggressive treatment the median survival after diagnosis is approximately 12 months. Chemotherapy with the nitrosourea BCNU, the methylating agents procarbazine or temozolomide, and other alkylating agents is effective and can prolong survival but the therapeutic benefit is attenuated due to hematopoietic toxicity which limits dose-escalation of these drugs. We have previously demonstrated in the dog and non-human primate model efficient gene marking, in vivo selection and chemoprotection from temozolomide and BCNU following transplantation with MGMTP140K gene-modified cells. Thus, we wanted to explore the efficacy of autologous transplantation in macaques and baboons using cells gene-modified with an MLV-based gammaretrovirus vector developed for a pending clinical trial. This retroviral vector contains a myeloproliferative sarcoma virus LTR, negative control region deleted, dl587rev primer binding site (MND) vector backbone, which expresses MGMTP140K from the 5′ LTR promoter, and is pseudotyped with the gibbon ape leukemia virus (GALV) envelope produced from Phoenix-GALV packaging cells (PhGALV-MND.GRS.P140K c38). Following transduction the gene marking in pre-infusion colony forming units (CFUs) was 74.3% and 69.8% in the macaque and baboon respectively as determined by CFU-PCR. Intracellular MGMT-staining of cultured baboon cells 4 (76.4%) and 11 (89.9%) days after transduction confirmed high gene transfer levels. Both animals recovered neutrophil and platelet levels within expected time frames relative to historical controls and the average provirus copy number determined by real-time PCR approximately one month after transplantation was 0.14 and 0.72 in the macaque and baboon respectively. Retrovirus integration site analysis in the macaque 90 days after transplantation, and before chemotherapy, confirmed polyclonal hematopoietic reconstitution. There was no indication of progression to a pre-leukemic state. The macaque has been treated twice with O6-benzylguanine (O6BG) and BCNU and the gene marking has stably increased approximately 2.5-fold. Aside from transient elevated liver enzymes following O6BG/BCNU treatment no additional extra-hematopoietic toxicity has been observed. In summary, we have been able to achieve efficient polyclonal gene marking with MGMTP140K gene-modified cells using a vector designed for clinical application in both macaques and baboons and have preliminary evidence of in vivo selection in the macaque. We believe that these large animal studies closely reflect a clinical setting and will help to further improve clinical HSC gene therapy.

Description: Abstract 3572 Poster Board III-509 Strategies using gene-modified hematopoietic stem cells to treat various severe hematopoietic diseases, including but not limited to hemoglobinopathies, will likely require high levels of gene marking. Here we have established efficient and stable in vivo selection in nonhuman primates using methylguanine methyltransferase (MGMTP140K). In the macaque (Macaca nemestrina) we were able to increase pre-chemotherapy lentiviral gene marking levels of 11.3% in granulocytes and 15.3% in lymphocytes to a post-chemotherapy gene marking level of 76.9% in granulocytes and 49.0% in lymphocytes. Furthermore, stable increases in gene marking were also observed in red blood cells (RBCs) and platelets (PLTs) with a pre-chemotherapy gene marking level of 5.6% and 6.7%, respectively, and a post-chemotherapy gene marking level of 15.2% and 64.0%, respectively. Importantly, the chemotherapy regimen was well tolerated, and engraftment was polyclonal as determined by analyzing long-term repopulating clones by LAM-PCR. In order to minimize extra-hematopoietic toxicity we have began to test a more clinically applicable conditioning regimen in the macaque model. This reduced intensity conditioning regimen should allow treatment of patients with severe hematopoietic or infectious diseases, who may not tolerate a high dose conditioning regimen. We tested targeted busulfan for conditioning to provide sufficient myelosuppression and to facilitate engraftment of chemoprotected hematopoietic stem cells while minimizing extra-hematopoietic toxicity. Following conditioning with busulfan (4 mg/kg/day for 2 days) and infusion of gene modified cells (∼1.7 × 107 CD34-selected cells/kg), there was moderate cytopenia with ANC

Description: AIDS remains a significant health problem worldwide despite the advent of highly active antiretroviral therapy (HAART). Although substantial efforts have been made to develop a vaccine there is still no cure and alternative strategies are needed to treat HIV infection and to control its spread. Our goal is to evaluate lentiviral vectors that inhibit HIV replication by RNA interference (RNAi) in a non-human primate SHIV model to develop a hematopoietic stem cell (HSC) gene therapy for AIDS. SHIV89.6 P is a chimeric virus comprised of an SIV genome that contains the tat, rev and env genes of HIV and infects both T lymphocytes and macrophages. Infection of non-human primates with SHIV89.6P results in significant decreases in CD4+ T cells as early as 4 weeks post infection, and is currently the best large animal model available to test gene therapy strategies for AIDS. We present here data showing efficient transduction of M. nemestrina CD34+ cells with an HIV-based lentiviral vector and RNAi-mediated inhibition of SHIV89.6 P replication in a hybrid T/B lymphocyte cell line (CEMx174). Although others reported a block to transduction of M. mulatta CD34+ cells with an HIV-based lentiviral vector, we observed efficient transduction rates (» 50%) of M. nemestrina CD34+ cells, comparable to transduction rates observed in human CD34+ cells (» 60%). To determine effectiveness of anti tat/rev shRNA to inhibit SHIV89.6P in vitro, a human T cell/B cell hybrid cell line (CEMx174) was transduced with a lentiviral vector expressing a short-hairpin RNA (shRNA) targeted to both HIV tat and rev sequences that also contained either a GFP reporter gene or a MGMT(G156A) resistance gene at MOIs of 1.3 and 3 respectively. Polyclonal populations of CEMx174 cells transduced with the GFP and MGMT(G156A) vectors were challenged with a 2.15x103 TCID50 dose of SHIV 89.6P. One week post challenge, expression of both tat and rev transcripts was reduced 88% and 97% respectively in these cultures as measured by real-time PCR. In summary, we have shown efficient HIV-based lentiviral transduction of M. nemestrina cells and efficient inhibition of SHIV infection by shRNA against HIV tat and rev thus providing a useful model to test lentiviral-mediated anti-HIV RNAi stem cell gene therapy in vivo.

Description: In vivo gene therapy has several benefits over ex vivo hematopoietic stem cell gene therapy, including the correction of progenitor cells in their native environments, the portability of the treatment to the patient, and the ability to administer serial doses of therapeutic vector. Foamy viruses (FV) are ideal vectors for in vivo gene therapy because they are non-pathogenic in humans, they exhibit increased serum stability and they integrate into host genomes with a favorable integration pattern. We recently demonstrated that intravenous injection of a FV vector expressing the human common gamma chain (γC) under the constitutively active short elongation factor 1α (EF1α) promoter is sufficient to drive development of functional CD3+ lymphocytes in canine X-SCID (Burtner CR et al. Intravenous injection of a foamy virus vector to correct canine SCID-X1. Blood. 2014;123(23):3578-84). However, retroviral integration site analysis in that study indicated that T cell reconstitution occurred through the correction of a limited number of progenitors, possibly due to sub-therapeutic expression levels from the EF1α promoter. To address this issue, we are evaluating multiple parameters of vector design for in vivo gene therapy that include different promoters and different fluorophores. We performed a head-to-head comparison of two promoters, our previously used EF1α promoter and the human phosphoglycerate kinase (PGK) promoter, by simultaneously injecting three X-SCID pups with equal titers of two therapeutic, human γC-encoding FV vectors. These vectors expressed the fluorophores GFP or mCherry to allow for tracking of transduced cells. Each dog received between 3 and 4 x 108 infectious units of each FV vector. In all treated dogs, lymphocyte marking in the PGK arm reached 50% between day 60 and day 110 post-injection and continued to expand over time, while the EF1α arm peaked at day 42 and never expanded above 10% (Fig 1A). Interestingly, the expansion of T lymphocytes from gene-modified cells expressing γC under the PGK promoter appeared to preclude further development of T cells by the EF1α arm, suggesting competition within the expanding T cell niche. The development of total CD3+ T cells achieved therapeutic levels (1000 cells/μL of blood) in all three dogs between day 70 and day 130 post-treatment (Fig 1B). We further validated the functionality of these cells by showing that they express a diverse T cell receptor repertoire using spectratyping analysis. In addition, peripheral blood mononuclear cells from the treated animals could be activated in vitro by exposure to the mitogen Phytohemagglutinin A at a level comparable to normal cells. Immunization of the treated dogs with bacteriophage ΦX174 showed production of specific IgG antibodies, suggesting the ability of B lymphocytes to undergo isotype switching. Finally, retroviral integration site analysis revealed polyclonal contribution to the reconstituting T cells. In summary, our data suggest that the PGK promoter results in a robust and sustained correction of progenitor T cells in a relevant large-animal disease model for primary immunodeficiency. The outcome in dogs was substantially improved compared to our previous study using EF1α, where robust lymphocyte marking was achieved in only 2 of 5 dogs, and where clonal dominance was observed. Ongoing work will determine whether the superior performance of the PGK vector is due to higher γC expression in PGK vs. EF1α corrected cells. Figure 1. T-cells expansion in X-SCID dogs following FV treatment. A) Percent of gene-modified peripheral blood lymphocytes in each experimental arm after in vivo gene therapy. B) Absolute CD3+ count per μL peripheral blood in all treated animals. Dotted line indicates therapeutic counts of CD3+ cells. Figure 1. T-cells expansion in X-SCID dogs following FV treatment. A) Percent of gene-modified peripheral blood lymphocytes in each experimental arm after in vivo gene therapy. B) Absolute CD3+ count per μL peripheral blood in all treated animals. Dotted line indicates therapeutic counts of CD3+ cells. Disclosures No relevant conflicts of interest to declare.

Description: AIDS remains a significant health problem worldwide despite the advent of highly active antiretroviral therapy (HAART). Although substantial efforts have been made to develop a vaccine there is still no cure and alternative strategies are needed to treat HIV infection and to control its spread. Our goal is to evaluate lenti and foamy retroviral vectors that inhibit HIV replication by RNAi in a non-human primate SHIV model to develop a hematopoietic stem cell (HSC) gene therapy for AIDS. SHIV is a chimeric virus comprised of an SIV genome that contains the tat, rev and env genes of HIV and infects both T lymphocytes and macrophages. Infection of non-human primates with SHIV results in significant decreases in CD4+ T cells as early as 4 weeks post infection, and is currently the best large animal model available to test gene therapy strategies for AIDS. However inefficient gene delivery to hematopoietic stem cells has limited progress for AIDS gene therapy. We have developed both lenti and foamy retroviral vectors that contain methylguanine-DNA-methyltransferase (MGMT) expression cassettes to allow for in vivo selection, and have transduced macaque (M. nemestrina) long term repopulating cells with both vector systems. Following transplantation we observed rapid engraftment and levels of gene marking in the peripheral blood that should allow us to in vivo select both lenti and foamy-marked hematopoietic repopulating cells. In one animal transplanted with a lentiviral vector we obtained marking at 265 days post-transplant of over 30% in peripheral blood granulocytes and 20% in peripheral blood lymphocytes prior to in vivo selection. Anti-SHIV/HIV transgene cassettes targeting tat and rev that allow for potent inhibition of SHIV and HIV replication in vitro have been incorporated into both lenti and foamy vectors and we have transduced macaque long term repopulating cells with lenti vectors containing an anti-HIV cassette. We are currently developing protocols for efficient in vivo selection and future studies will investigate the ability of macaque hematopoietic repopulating cells transduced with lenti and foamy MGMT anti-HIV vectors to inhibit SHIV infection ex vivo and in vivo.

Description: Retroviral vectors have been the most effective gene delivery vehicles for hematopoietic stem cell (HSC) gene therapy and patients are now being successfully treated for genetic diseases. Following the success of gene therapy for inherited disorders, such as X-linked severe combined immunodeficiency (SCID), three of the patients developed overt leukemia, and a spontaneous expansion of gene-marked cells has been described in two patients treated in an X-linked chronic granulomatous clinical trial. In spite of these outcomes clinical gene therapy trials continue to show efficacy for patients with genetic diseases. Valuable data has been generated regarding retrovirus integration profiles and factors that can lead to clonal dominance (i.e. enhancer activation and multiple integration sites) in murine models. Large animal studies extend these analyses to clinically predictive models, that should more accurately indicate what would occur in human clinical trials. We analyzed the retrovirus integration profile in dogs transplanted with cells gene-modified with either gammaretrovirus (n=5 dogs), HIV-derived lentivirus (n=6 dogs), or foamy virus (n=2 dogs) vectors using a sensitive LAM-PCR method modified to facilitate high-throughput analysis. The samples used for LAM-PCR ranged from 95–600 days after transplantation. We analyzed over 14,500 sequence reads and were able to unambiguously align a total of 555 unique integration sites to the dog genome with 82 unique gammaretroviral integrants, 210 unique lentiviral integrants, and 263 unique foamy viral integrants. We defined the integration patterns relative to transcription units, a subset of previously defined proto-oncogenes and CpG islands. The most prevalent clustering within 50 kilobases of RefSeq transcription start sites and into CpG islands was seen in gammaretroviral integrants (73.2% and 3.7%, respectively) and to a lesser extent in foamy viral integrants (53.6% and 3.4%, respectively). Regarding integration into RefSeq genes, lentiviral integrants showed the most significant increase (58.6%) relative to random integration analysis (37.3%). Gammaretroviral integrants were also significantly increased both in proto-oncogenes (7.3%) and within 50 kilobases of the transcription start site of proto-oncogenes (7.3%). In addition, even though fewer sites have been analyzed and localized for gammaretrovirus vectors, compared to lentiviral and foamy viral sites, we found two distinct gammaretroviral integrants in the MDS/Evi1 locus with no lentiviral or foamy viral integrants localized in this region. While no single factor distinguishes one of the retroviruses as the ‘safest’ vector system, the low frequency of integration of foamy virus into RefSeq genes and the decreased density of lentiviral integrants around transcription start sites suggest that these vectors may be preferred relative to gammaretroviral vectors. Additionally, high titer self-inactivating (SIN) vector design has been achieved using both lentivirus and foamy virus which should decrease the risk of enhancer activation relative to intact LTR constructs. These aspects and others, including weaker/regulated internal promoters, and chromatin insulators are important factors that should be considered when designing vectors for clinical gene therapy applications.

Description: HOXB4 is considered to be the only HOX gene that promotes self-renewal of hematopoietic stem cells without causing leukemia in mouse models. We investigated whether HOXB4 overexpression has similar effects in a clinically relevant canine model. A competitive repopulation assay was performed in three dogs in which CD34+ cells were transduced with MSCV-based gammaretroviral vectors expressing HOXB4GFP or control YFP. We observed up to 4-fold higher marking levels in granulocytes for the HOXB4GFP arm relative to the control 1 month after transplantation. The marking levels eventually decreased in all three animals and two dogs (G374, G450) have now been followed for more than 18 months. In G374, the marking levels for both arms stabilized at ~2% after 2 months post-transplantation. Between 14 and 20 months post-transplantation, the HOXB4GFP marking steadily increased to 〉95%, while YFP marking decreased to 0.1%. G374 was euthanized 21 months after transplantation due to declining health. Flow cytometry analysis showed that ~50% of BM cells expressed the monocyte marker CD14 and ~8% expressed the granulocyte marker DM5, all of which also expressed HOXB4GFP. CD3 and CD21 were expressed in 2% and 1% of cells, respectively, but these cells did not express HOXB4GFP. Bone marrow necropsy demonstrated significantly increased numbers of blast cells, consistent with a myelomonocytic leukemia. Southern blot analyses of G374 BM and PB samples identified 2 bands with the same intensity, suggesting a single dominant clone with 2 integration sites. LAM-PCR analysis identified two vector proviruses integrated ~100 kb upstream of c-myb, and into intron 3 of PRDM16. Western blot analysis confirmed expression of HOXB4 in cultured G374 BM cells but the levels of c-myb in these cells were not different from control HOXB4-transduced BM cells as determined by RT-PCR. The expression of PRDM16 exons 1–3 was not detected in cells from dog G374 or in control cells, however, PRDM16 exon 4 was expressed in G374 cells but not in control cells. RT-PCR using primers located in the MSCV LTR and in PRDM16 exon 4 identified a unique band and sequencing of this product showed that the 5′ LTR was spliced with PRDM16 exon 4 creating a short PRDM16 isoform which has been observed in human leukemias. These data suggest that HOXB4 overexpression in collaboration with integration-induced activation of PRDM16 led to the leukemia. Southern blot and SYBR green Q-PCR showed that the leukemic clone contributed to ~20% hematopoiesis in BM 6 months after transplantation, and gradually decreased to ~2% before final expansion of the clone, suggesting accumulation of other mutation(s) were required for overt leukemia. Karyotype analysis of BM cells has not shown any major abnormalities but we are currently performing analyses to search for minor abnormalities such as gene duplications and deletions. Recently, HOXB4GFP marking in dog G450 PB and BM has increased to 20% and 80%, respectively, while YFP marking has decreased to ~1%. Southern blot analysis has identified a single dominant band and a BM biopsy showed substantially increased blast cells. Of note, we have not observed leukemia in 〉30 dogs followed long term that received transduced cells without HOXB4. In summary, HOXB4 overexpression together with insertional mutagenesis by virus integration has induced leukemia in the canine model, demonstrating the utility of this model to study the safety of gene therapy.

Description: In vivo gene therapy has several benefits over ex vivo hematopoietic stem cell gene therapy, including the correction of progenitor cells in their native environments, the portability of the treatment to the patient, and the ability to administer serial doses of therapeutic vector. Foamy viruses (FV) are ideal vectors for in vivo gene therapy for 3 primary reasons: (1) FV are non-pathogenic in humans, (2) they exhibit enhanced serum stability as compared to lentiviruses packaged with the vesicular stomatitis virus glycoprotein (VSV-G), and (3) FV integrate into host genomes with a favorable integration pattern. We recently demonstrated that intravenous injection of a FV vector expressing the human common gamma chain (γC) under the constitutively active short elongation factor 1α (EF1α) promoter is sufficient to drive development of CD3+ lymphocytes in canine X-SCID, which undergo T cell receptor rearrangement and exhibit a functional signaling response to T cell activating mitogens (Burtner CR, Beard BC, Kennedy DR, et al. Intravenous injection of a foamy virus vector to correct canine SCID-X1. Blood. 2014;123(23):3578-84). However, retroviral integration site analysis in that study indicated that T cell reconstitution occurred through the correction of a limited number of progenitors, possibly due to sub-therapeutic expression levels from the EF1α promoter. To address this issue, we are evaluating multiple parameters of vector design for in vivo gene therapy, including different promoters, using injections of vectors marked with different fluorophores. Preliminary data indicated that ex vivo transduction of canine CD34+ cells with a FV vector expressing human γC and a fluorescent reporter under the human phosphoglycerate kinase (PGK) promoter resulted in higher transduction efficiencies and increased mean fluorescence intensity, compared to that of an identical vector containing the EF1α promoter. We therefore performed a head-to-head comparison of the two promoters by simultaneously injecting X-SCID pups with equal titers of 2 therapeutic, human γC-encoding FV vectors that differed only in the promoter used to drive human γC expression and in the fluorophore color to distinguish gene-marked cells (GFP and mCherry). Each dog received 4 x 108 infectious units of each FV vector. A significant population of gene-marked lymphocytes appeared in the PGK arm 42 days post in vivo gene therapy, which continued to expand over the next two months of follow-up (Fig 1A). By 84 days post injection, lymphocyte gene marking in the competitive PGK arm reached 60% in both dogs. For comparison, this robust level of lymphocyte gene marking was achieved in only 2 of 5 dogs after 122 and 160 days, respectively, in our previous EF1α virus treated cohort. In contrast, the EF1α arm peaked at 42 days after in vivo gene therapy and never expanded above 10% (Fig 1A). Interestingly, the expansion of T lymphocytes from gene-modified cells expressing γC under the PGK promoter appeared to preclude further development of T cells by the by the EF1α arm, suggesting competition within the expanding T cell niche. The expansion of gene-marked lymphocytes was followed by the development of CD3+ T cells, leading to a therapeutic level of CD3+ cells (1000 cells/μl of blood) in both dogs (Fig 1B). Additionally, our data indicate low but persistent gene marking in other blood cells, including granulocytes and B cells, with B cell marking in one animal exceeding 2% in the PGK arm. Our data suggest that the PGK promoter results in a robust and sustained correction of progenitor T cells in a relevant large-animal disease model for primary immunodeficiency. These data also highlight the utility of the in vivo approach to explore key parameters of vector design in competitive repopulation experiments that may be useful for other diseases. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Gene transfer into hematopoietic stem cells (HSCs) is an ideal treatment strategy for many genetic and hematologic diseases. However, progress has been limited by the low HSC transduction rates obtained with retroviral vectors based on murine leukemia viruses. This study examined the potential of vectors derived from the nonpathogenic human foamy virus (HFV) to transduce human CD34+ cells and murine HSCs. More than 80% of human hematopoietic progenitors present in CD34+ cell preparations derived from cord blood were transduced by a single overnight exposure to HFV vector stocks. Mice that received transduced bone marrow cells expressed the vector-encoded transgene long term in all major hematopoietic cell lineages and in over 50% of cells in some animals. Secondary bone marrow transplants and integration site analysis confirmed that gene transfer occurred at the stem cell level. Transgene silencing was not observed. Thus vectors based on foamy viruses represent a promising approach for HSC gene therapy.

Description: Key PointsIntravenous injection of a foamy virus carrying a corrective gene facilitates immune cell development in a canine model of SCID-X1. Integration site analysis revealed polyclonal reconstitution in all dogs with evidence for clonal dominance in at least 1 time point.