Retroviral Integration Site Analysis Research Articles

HSCs Engraft in a Stochastic Pattern and Form Clonal Pools after Transplantation

Hematopoietic stem cells (HSCs) are considered the only cell type capable of self-renewing and generating all blood lineages. HSCs are further assumed to be rare, barely proliferating, and not contributing to short-term hematopoietic recovery after transplantation. Although widely accepted, individual definitions have been challenged. Tracking of murine HSCs showed continuous contribution during steady-state hematopoiesis. Similarly, single HSCs can reconstitute the entire murine host illustrating extensive proliferative capabilities without immediate exhaustion while maintaining long-term persistence. Studying these findings in humans is challenging. Knowledge about the engraftment kinetics and contribution of human HSCs during recovery is limited to clinical trials of gene therapy patients enrolled in clonal tracking studies using retroviral integration site analysis (ISA). However, the tracking of human HSCs remains under-investigated due to the limited number of specimens possible to obtain, especially during the early phases of reconstitution where blood cell counts are low. In addition, clone tracking is impacted by technical limitations. While ISA is reliable to monitor the potential outgrowth of dominant clones, low sensitivity and high error rates require significant data exclusion and sophisticated statistical tests to ensure data reliability (Adair et al. 2020, Molecular Therapy MCD). Lack of sensitivity and loss of low abundance clones can be overcome by increasing the frequency of sampling (high density) as well as repeated sampling, aspects hard to achieve in patients. To investigate the clonal dynamics in the early phases of hematopoietic reconstitution and determine the onset of HSC contribution, we performed high-density sampling for ISA in nonhuman primates (NHPs). In the first month of hematopoietic recovery, weekly blood samples were taken to enhance data density. We applied a new clonal tracking approach to reliably detect the contribution of multipotent long-term persisting HSCs during neutrophil recovery even in samples with low cellularity during early recovery. Animals were followed for 4.5 years to collect HSC clone signatures, and multipotent stem cell clones were retrospectively identified in early samples. Finally, observed changes in clonal diversity in vivo were used to inform a simulation of hematopoietic reconstitution to determine the temporal involvement of HSCs. Contribution of multipotent long-term persisting HSCs clones was detected in the very first blood samples taken 2 to 3 weeks post-transplant during neutrophil recovery. HSCs became the dominant source for mature blood cells in the peripheral blood as early as 50 days post-transplant. Based on these finding, simulations predicted that hematopoietic recovery is primarily HSC driven, differentiating HSC clones are the main source of blood production during recovery, and that long-term persisting HSC clones expand forming clonal pools. The ability of HSCs to symmetrically expand was confirmed ex vivo in colony-forming cells assays as well as in vivo using ISA and tracking of expressed barcodes. As predicted by the simulation, we observed a rapid decline of clonal diversity and individual HSCs symmetrically expand forming clonal pools. Here, we show evidence that long-term persisting multipotent HSCs actively contribute during early hematopoietic reconstitution after transplantation. Enhanced sampling showed that multipotent HSCs produce neutrophils during recovery, are the main source of mature blood cells as early as 50 days post-transplant, and symmetrically divide to form clonal pools long-term. Most importantly, observed changes in the clonal diversity during early recovery suggest a stochastic HSC engraftment and differentiation pattern rather than a bi-phasic reconstitution initially driven by short-term progenitors. Early blood production is driven by these HSC clones that are not predetermined but stochastically contribute to a limited number of lineages before they disappear - a population of cells previously incorrectly identified as lineage-restricted short-term engrafting progenitors. Consequently, we here propose a revised model of hematopoietic reconstitution and provide novel insights into HSC biology with direct implications for the design of HSC transplantation as well as gene therapy studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High-Density Clonal Analysis Reveals Highly Active Contribution of Multipotent Hematopoietic Stem Cells during Early Phases of Hematopoietic Recovery after Transplantation

Recovery after conditioning and transplantation of hematopoietic stem and progenitor cells (HSPC) is thought to be biphasic, with short-term engrafting progenitors driving the recovery for 6-9 months and multipotent hematopoietic stem cells (HSCs) providing long-term repopulation. Recent clonal tracking data from autologous human gene therapy trials seems to support this model (Biasco et al. 2016, Cell Stem Cell; Six et al. 2020, Blood). These recent reports investigating the contribution of HSCs in patients are based on the longitudinal tracking of thousands of gene-marked cells using retroviral integration site analysis (ISA). While this technology is very reliable to follow gene therapy patients and monitor the potential outgrowth of dominant or malignant clones, low sensitivity and high error rates require significant data exclusion and sophisticated statistical tests to ensure data reliability (Adair et al. 2020, Molecular Therapy MCD). Lack of sensitivity can be overcome by increasing the frequency (high density) of sampling. However, limited material from patients remains a bottleneck for improved data quality and, consequently, correct interpretation of such complex datasets.To overcome the limitations of ISA and determine the onset of HSC contribution we performed high-density sampling for ISA in nonhuman primates (NHPs) transplanted with gene-modified HSCs. In the first month of hematopoietic recovery weekly blood samples were taken to enhance data density and increase the reliablity to detect clones with low abundance. Animals were followed up to 5 years to confirm that identified HSC clones persist long-term. Finally, clonal tracking data from the NHPs was used to inform a simulation of hematopoietic reconstitution, determine the temporal involvement of HSCs, and refine the phases of hematopoietic recovery after myeloablation and HSC transplantation.In contrast to the current biphasic model, contribution of multipotent HSCs clones was detected in the very first blood samples taken 2 to 3 weeks post-transplant during neutrophil recovery. HSC clones found in these early time points persisted long-term throughout the entire follow-up and were detected in bone marrow CD34 + cells 4 years later. Most surprisingly, multipotent HSCs became the dominant source for mature blood cells in the peripheral blood as early as 50 days post-transplant. To understand the observed kinetics of HSC contribution and change in clonal diversity in our dataset, we simulated the clonal outgrowth and differentiation of multipotent clones. Simulations predicted that hematopoietic recovery is primarily HSC driven and HSC contribution follows a stochastic pattern. Finally, to confirm that HSCs proliferation and differentiation is a stochastic process, in vitro experiments in colony-forming cell (CFC) assays were carried out. As predicted, the decision of individual HSCs to either grow into a larger pool or differentiate and get lost followed the same kinetics as observed in vivo.Here, we show evidence that long-term persisting multipotent HSCs actively contribute during early hematopoietic reconstitution after myeloablation and HSC transplantation. Enhanced sampling showed that multipotent HSCs produce neutrophils during recovery and become the predominant source of mature blood cells as early as 50 days post-transplant. Most importantly, observed changes in the clonal diversity during early recovery suggest a stochastic engraftment of HSCs rather than a bi-phasic reconstitution initially driven by short-term progenitors. These findings should have important implications for the design of ex vivo and in vivo HSC gene therapy and genome editing approaches. [Display omitted] DisclosuresRadtke: 47 Inc.: Consultancy; Ensoma Inc.: Consultancy. Kiem: Homology Medicines: Consultancy; VOR Biopharma: Consultancy; Ensoma Inc.: Consultancy, Current holder of individual stocks in a privately-held company.

A Lentiviral Fluorescent Genetic Barcoding System for Flow Cytometry-Based Multiplex Tracking

Retroviral integration site analysis and barcoding have been instrumental for multiplex clonal fate mapping, although their use imposes an inherent delay between sample acquisition and data analysis. Monitoring of multiple cell populations in real time would be advantageous, but multiplex assays compatible with flow cytometric tracking of competitive growth behavior are currently limited. We here describe the development and initial validation of three generations of lentiviral fluorescent genetic barcoding (FGB) systems that allow the creation of 26, 14, or 6 unique labels. Color-coded populations could be tracked in multiplex invitro assays for up to 28days by flow cytometry using all three vector systems. Those involving lower levels of multiplexing eased color-code generation and the reliability of vector expression and enabled functional invitro and invivo studies. In proof-of-principle experiments, FGB vectors facilitated invitro multiplex screening of microRNA (miRNA)-induced growth advantages, as well as the invivo recovery of color-coded progeny of murine and human hematopoietic stem cells. This novel series of FGB vectors provides new tools for assessing comparative growth properties in invitro and invivo multiplexing experiments, while simultaneously allowing for a reduction in sample numbers by up to 26-fold.

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.

Pgk-Mediated Expression of Common Gamma Chain Is More Effective Than EF1a for Therapeutic Immune Reconstitution of X-SCID Dogs after In Vivo Gene Therapy with Foamy Virus Vector

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. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

528. Generation of a Cocal Envelope Packaging Cell Line for Robust Lentiviral Gene Transfer Into Hematopoietic Stem Cells and T Cells

Lentiviral vectors (LVs) are routinely used for stable gene transfer and have demonstrated great promise in clinical hematopoietic stem cell and immune cell gene therapy. LVs are commonly pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G), which confers broad tropism to the vector and allows for vector concentration by centrifugation. However, the use of VSV-G has several limitations, such as susceptibility to inactivation by human serum complement making it unsuitable for in vivo delivery, and toxicity when constitutively expressed, which has impeded efforts to generate stable producer cell lines. With the goal to generate a self-inactivating LV packaging cell line, we stably expressed either the VSV-G or cocal envelope in HEK293T cells along with other required helper genes, 3rd generation gagpol and rev. Genes were sequentially introduced in cells by co-transfection with plasmids containing an antibiotic resistance gene. After selection, best producer clones were isolated by limiting dilution and were treated with the histone deacetylase inhibitor sodium butyrate to enhance vector titer. The resulting cocal packaging cell line produces 20 to 50-fold more infectious particles as compared to VSV-G expressing cells, reaching titers averaging 108 infectious units/mL upon concentration. We also extend on our previous studies (Trobridge, 2010) by showing more robust gene transfer for cocal-pseudotyped LVs as compared to VSV-G pseudotyped LVs in human (81% cocal vs. 45% VSV-G, MOI 5) and non-human primate (Macaca nemestrina) CD34+ HSPCs (45% cocal vs. 18% VSV-G, MOI 2×5) while preserving similar differentiation potential as determined by CFC assays. Cocal vectors also transduced human CD3+ blood cells more efficiently than VSV-G vectors (65% cocal vs. 42% VSV-G vs., MOI 3) and showed comparable transduction efficiency in non-human primate CD3+ cells. Using a competitive repopulation approach in the non-human primate model, we find increased gene marking in repopulating cells transduced with a cocal pseudotyped LVs compared to repopulating cells transduced with the same lentivirus backbone pseudotyped with VSV-G. We observed a dramatic difference in the granulocyte fraction where marking with the cocal vector reaches ~90% at 70 days post transplantation. Retroviral integration site analysis is currently underway to characterize the integration profile of cocal vs. VSV-G modified clones. Overall, our study suggests that the cocal envelope outperforms the VSV-G envelope in a stable lentivirus packaging cell line, which may prove useful in current gene therapy efforts to generate large-scale clinical grade vectors.

Robust Therapeutic Expression of the Common Gamma Chain with the Human Pgk Promoter Using Foamy Virus in Vivo Gene Therapy in a Canine Model of Severe Combined Immunodeficiency

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. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Intravenous injection of a foamy virus vector to correct canine SCID-X1

AbstractCurrent approaches to hematopoietic stem cell (HSC) gene therapy involve the collection and ex vivo manipulation of HSCs, a process associated with loss of stem cell multipotency and engraftment potential. An alternative approach for correcting blood-related diseases is the direct intravenous administration of viral vectors, so-called in vivo gene therapy. In this study, we evaluated the safety and efficacy of in vivo gene therapy using a foamy virus vector for the correction of canine X-linked severe combined immunodeficiency (SCID-X1). In newborn SCID-X1 dogs, injection of a foamy virus vector expressing the human IL2RG gene resulted in an expansion of lymphocytes expressing the common γ chain and the development of CD3+ T lymphocytes. CD3+ cells expressed CD4 and CD8 coreceptors, underwent antigen receptor gene rearrangement, and demonstrated functional maturity in response to T-cell mitogens. Retroviral integration site analysis in 4 animals revealed a polyclonal pattern of integration in all dogs with evidence for dominant clones. These results demonstrate that a foamy virus vector can be administered with therapeutic benefit in the SCID-X1 dog, a clinically relevant preclinical model for in vivo gene therapy.

No evidence of clonal dominance after transplant of HOXB4-expanded cord blood cells in a nonhuman primate model

Umbilical cord blood transplant continues to increase in prevalence as a treatment option for various hematopoietic and immune disorders. Because of the limited number of cells available in a single cord blood unit, investigators have explored methods of increasing cell dose before transplant, including overexpression of the homeobox B4 (HOXB4) transcription factor. We have previously reported the development of leukemia in several nonhuman primate (NHP) subjects transplanted with HOXB4-expanded bone marrow cells at approximately 2 years posttransplant. Here, we provide long-term data for a NHP receiving a HOXB4-expanded cord blood graft. Longitudinal follow-up included gene marking analysis, complete blood counts, morphologic/pathologic assessment, phenotypic analysis of subsets, and retroviral integration site analysis. In each of these independent assays, we saw no indication of clonal dominance, and all signs pointed toward normal, healthy hematopoiesis. Furthermore, in-depth clonal analysis of an animal that developed leukemia after transplantation of HOXB4-modified bone marrow cells showed that dominant clones could be detected as early as 6 months posttransplant using the genomic analysis technique detailed here. Parallel analysis of the cord blood transplant macaque showed no such sites. These findings demonstrate the ability to study the use of gene-modified and expanded cord blood cells in a NHP model.

Identification Of Genes/Signaling Pathways That Predispose To Therapy Related MDS (t-MDS)

Myelodysplastic syndrome (MDS) constitutes a complex set of hematopoietic stem cell diseases that involves defective differentiation into either one, some or all of the hematopoietic lineages. MDS is caused due to the accumulation of mutations in hematopoietic stem/progenitor cells (HSPCs). Therapy related MDS (t-MDS) is a secondary complication associated with prior chemo-radiation therapy for either cancer or non-cancer related diseases and accounts about 20% of the total MDS cases. Genes predisposing t-MDS in response to chemo-radiation therapy remain enigmatic. We combined a retroviral insertional mutagenesis approach with a radiation treatment regimen in the mouse in an attempt to identify novel t-MDS predisposing genes and signaling pathways. To this end, C57BL/6 low-density bone marrow (LDBM) cells were transduced with the SFβ91 EGFP+ retrovirus, which results in a very high level of insertional mutagenesis (dysregulation of genes in the vicinity of the integration site). Transduced LDBM cells were transplanted into BoyJ recipient mice. After confirming successful transplantation of donor cells into recipients, animals were treated 3-4 weeks post transplant with either 0, 0.5 or 2Gy radiation once a day for 3 days. Mice were monitored for aberrations in any of the hematopoietic lineages in both peripheral blood (PB) and bone marrow (BM, intra-femural aspirates) at frequent intervals thereafter. Mice irradiated with 2 Gy exhibited cytotoxic effects on different hematopoietic lineages 1-week post radiation. Currently at 35 weeks post-radiation, 4 mice treated with 0.5 Gy and 1 mouse treated with 2 Gy developed a tMDS/AML-like disease. In these animals, BM was saturated with transduced donor cells (measured by GFPpos cells, ranging from 82-97% in BM of different mice) and mice exhibited anemia, thrombocytopenia, splenomegaly and myeloid blasts in BM at different time points. Additionally, 1 mouse treated with 2 Gy developed T-cell leukemia with 95.5% GFPpos T cells in PB and lymphoid blasts in BM and 1 mouse with 98.7% GFPneg cells in BM, with 75% of contribution from Mac1/Gr1. Mice developing t-MDS/AML like disease were sacrificed and bone marrow (BM) cells were collected for analysis of retroviral integration sites. We performed ligation-mediated PCR (LM-PCR) using retroviral long terminal repeats (LTR) sequence based primers and amplifying into the mouse genomic region to identify the unique insertion sites. We observed unique band patterns for each of the four t-MDS/AML mice indicating that the clones driving the disease are probably unique, which were confirmed by sequencing of LM-PCR products to identify the genome integration sites. Further gene expression analysis will be performed on the genes located around the integration site to identify individual genes and/or signaling pathways that are altered in these mice. Validation of such genes or pathways in mice might facilitate the identification of novel genes that predispose to t-MDS/AML. Disclosures:No relevant conflicts of interest to declare.

Sox4 and CREB Cooperate in Myeloid Transformation,

Abstract 3452The cAMP Response Element Binding Protein, CREB, is a transcription factor that is critical for cell proliferation and survival in neuronal and hematopoietic cells. We previously reported that CREB is overexpressed in leukemic blasts from patients with AML. CREB overexpression is also associated with an increased risk of relapse and decreased event-free survival in AML patients. We generated transgenic mice in which CREB is expressed under the control of the myeloid specific promoter, hMRP8. In vitro, bone marrow progenitors from CREB transgenic mice have increased proliferative potential and replating ability and these mice develop myeloproliferative disease, but not AML. Therefore, CREB is not sufficient to fully transform hematopoietic cells. To identify genes that cooperate with CREB, we performed retroviral insertional mutagenesis with CREB or wildtype C57/Blk6 mice. Newborn mice were infected with the MOL4070LTR retrovirus. Leukemia was observed in both CREB transgenic and wild type mice, however, latency for disease was significantly shortened in CREB transgenic mice at 9 months vs. 14 months for wild type mice (p<0.001). Infected mice developed hepatosplenomegaly (weighing up to 10-fold more than spleens from uninfected wild type mice). The leukemic phenotype by FACs analysis showed that 75% (24/32 mice analyzed) had AML with blast cells expressing Gr-1/Mac-1. Therefore, wild type MOL4070LTR infected mice had decreased penetrance for myeloid disease (45% or 5/11 mice analyzed). To identify potential cooperating oncogenes, genomic DNA was subjected to long-mediated PCR analysis to amplify genomic sequences upstream of the virus. Viral integration sites were identified by Southern blot analysis with genomic DNA from spleens from mice with leukemia. These sequences were mapped to the mouse genome to identify the chromosomal location. Blast analysis of retroviral integration sites (RIS) was performed on the publicly available Ensembl database and compared to known common integration sites on the Retroviral Tagged Cancer Gene Database (RTCGD http://rtcgd.ncifcrf.gov/). Sequencing of integration sites identified previously known oncogenes, such as Evi1/Mds, Evi5/Gfi1, Myb/Ahi, and Ras. Common integration sites identified in multiple mice include, sox4, Evi5/Gfi1, Myb/Ahi, Cbfa2t3. The highest incidence of integration in the mutagenesis screen was in the sox4 gene. Sox4 is a transcription factor that regulates embryonic development and cell fate. Transduction of CREB transgenic mouse bone marrow progenitor cells with a sox4 retrovirus increased survival and self-renewal by 2-fold (p<0.01) in serial replating and methylcellulose colony assays compared to wild type bone marrow progenitors. CREB overexpressing bone marrow progenitor cells stained with Brdu had a 10% increase in cycling cells compared to wild type bone marrow progenitor cells transduced with sox4 retrovirus (p<0.0001). Consistent with this observation is that sox4 retroviral transduction of CREB transgenic bone marrow cells led to increased CREB and cyclin D expression (p<0.01 and p<0.001, respectively). CREB transgenic mouse bone marrow transduced with sox4 retrovirus and stained with Annexin/7AAD also showed decreased apoptosis when cultured in low serum and absence of growth factor (p<0.05). Furthermore, the expression of the sox4 gene was increased in bone marrow cells from AML patients that also overexpressed CREB. Our results indicate that sox4 and CREB cooperate and contribute to transformation of hematopoietic progenitor cells. Disclosures:No relevant conflicts of interest to declare.

Acute Myeloid Leukemia in Mice Associated with Retrovirally-Mediated Overexpression of HOXB4

Numerous studies have established that retrovirally-mediated overexpression of the homeobox gene HOXB4 in murine bone marrow cells (BMCs) enhances expansion of hematopoietic stem cells (HSCs) in vivo and in vitro without causing hematopoietic abnormalities or impairing the ability of HSCs to give rise to differentiated progeny. More recently, however, retrovirally-mediated overexpression of HOXB4 in BMCs of large animals, such as dogs and macaques, has been implicated in leukemogenesis, but there are no prior reports of such a phenomenon occurring in mice transplanted with HOXB4-transduced BMCs. We report here two independent occurrences of acute myeloid leukemia (AML) in mice that received transplants of BMCs transduced with a HOXB4-overexpressing retrovirus. 5-FU treated BMCs were transduced with a MSCV-HOXB4-IRES-GFP retrovirus and transplanted into lethally irradiated mice. Two of the 54 mice (#1869 & #2050), transplanted with HOXB4-transduced BMCs, developed AML at 4 and 9.5 months, respectively, following transplantation. None of the 90 control mice, transplanted with BMCs transduced with the parent MSCV-IRES-GFP retrovirus, developed leukemia. Leukemia 1869 (L1) manifested marked leukocytosis, thrombocytopenia and mild anemia. Results of morphological and immunophenotyping analyses of bone marrow were consistent with AML. Leukemia 2050 (L2) also showed marked leukocytosis and significant anemia, but a near normal platelet count. Morphological analysis revealed a myelomonocytic phenotype, while immunophenotying showed the presence of multi-lineage cell surface markers, including myeloid (CD11b, Gr-1), erythroid (Ter119), and B lymphoid (B220) markers. A small percentage of cells that expressed the stem cell markers c-kit and Sca1 were identified in both leukemias. Both leukemias could be passaged by serial transplantation of leukemic bone marrow or spleen cells into wild type recipient mice. Analysis of retroviral integration sites identified ten separate integration sites in L1 and two in L2. This correlated with 6 fold higher levels of HOXB4 mRNA in L1 BMCs compared to L2 BMCs, as assessed by northern blot analysis that showed undetectable levels of HOXB4 mRNA in RNA of control BMCs. Some of the integration sites occurred in introns of genes known to be involved in leukemogenesis and resulted in aberrant expression of these genes. In L1, one of the retroviral integration sites occured in intron 1 of the N-ras gene and was associated with markedly increased levels of its mRNA in RNA of leukemic BMCs (15–20 fold higher levels compared to that in control BMCs). Another integration site in L1 was in intron 2 of the Lmo2 gene and was associated with a mild (~2 fold) increase in its mRNA level in leukemic cells. In L2, one of the retroviral integration sites was in intron 1 of the Prdm16 gene and was associated with markedly increased levels of its mRNA in leukemic cells. The second retroviral integration site in L2 was located ~50 kb upstream of the Notch1 gene and there was ~2 fold increase in its mRNA in BMCs of leukemic mice. We speculate that overexpression of HOXB4 acts in a cooperative or combinatorial fashion with overexpression of proto-oncogenes at the retroviral integration sites to induce AML in these mice. To test this hypothesis, we are testing various culture conditions to allow viable growth of the leukemic cells ex vivo and assess the effects of knockdown of HOXB4 mRNA accumulation on growth and differentiation of the cells in vitro, as well as on their ability to transmit leukemia in vivo. We are also characterizing the nature of the cells in the total leukemic cell population that are associated with in vivo leukemia-initiating activity. This is the first report of AML in mice associated with retrovirally-mediated overexpression of HOXB4.

Retroviral Integration Site Analysis and the Fate of Transduced Clones in anMDR1Gene Therapy Protocol Targeting Metastatic Breast Cancer

A clinical study of an MDR1 gene therapy protocol targeting metastatic breast cancer has been conducted in which the patients received high-dose chemotherapy, a transplant of MDR1-transduced autologous CD34(+) cells, and docetaxel. We herein report the molecular results of a 6-year follow-up of an individual in this study (patient 1). HaMDR-transduced cells, which had been initially detected in the peripheral blood of this individual, were found to have gradually decreased. After 10 cycles of docetaxel (days 71-316), MDR1 transgene levels were found to have increased, and then decreased to undetectable levels by day 1461. Thirty-eight MDR1-transduced clones were identified in patient 1, of which 11 showed a retroviral integration in close proximity to genes listed in the Retrovirus Tagged Cancer Gene Database (RTCGD). Four short-life clones in this group were found to harbor retroviral integrations close to the ZFHX1B, NOTCH1, BMI1, or HHEX gene; these genes have been frequently reported in the RTCGD. In addition, a long-lived RTCGD-hit clone, L-34, had a retroviral integration at a position 179 kb upstream of the EVI1 gene. L-34 was detectable on days 327-1154, but became undetectable 3 years after the docetaxel treatments had ceased. An additional three docetaxel-induced long-life clones showed comparable polymerase chain reaction profiles, which were also similar to that of the total MDR1-transduced cells. Our results thus show that docetaxel may have been effective in promoting the expansion of several MDR1-transduced clones in patient 1, but that they persist in the peripheral blood for only a few years.

Insertional Mutagenesis Reveals Progression Genes and Checkpoints in MYC/Runx2 Lymphomas

In this study, we have exploited the power of insertional mutagenesis to elucidate tumor progression pathways in mice carrying two oncogenes (MYC/Runx2) that collaborate to drive early lymphoma development. Neonatal infection of these mice with Moloney murine leukemia virus resulted in accelerated tumor onset with associated increases in clonal complexity and lymphoid dissemination. Large-scale analysis of retroviral integration sites in these tumors revealed a profound bias towards a narrow range of target genes, including Jdp2 (Jundm2), D cyclin, and Pim family genes. Remarkably, direct PCR analysis of integration hotspots revealed that every progressing tumor consisted of multiple clones harboring hits at these loci, giving access to large numbers of independent insertion events and uncovering the contrasting mutagenic mechanisms operating at each target gene. Direct PCR analysis showed that high-frequency targeting occurs only in the tumor environment in vivo and is specific for the progression gene set. These results indicate that early lymphomas in MYC/Runx2 mice remain dependent on exogenous growth signals, and that progression can be achieved by constitutive activation of pathways converging on a cell cycle checkpoint that acts as the major rate-limiting step for lymphoma outgrowth.

Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells

The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles. Cultured keratinocyte stem cells, known as holoclones, generate sheets of epithelium used to restore severe skin, mucosal and corneal defects. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder. Epidermal stem cells from an adult patient affected by LAM5-beta3-deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-beta3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up (1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease.

Retroviral integration site analysis identifies ICSBP as a collaborating tumor suppressor gene in NUP98-TOP1-induced leukemia

The NUP98-TOP1 fusion gene is one of 18 distinct translocations identified in acute myeloid leukemia involving the N-terminal portion of the nucleoporin NUP98. We previously reported that expression of NUP98-TOP in murine bone marrow induces a lethal, transplantable leukemia. However, the long latency suggests the in vivo acquisition of additional mutations and/or time required for clonal outgrowth of rare transformed cells arising from the collaboration of NUP98-TOP1 and a cooperating event. The aim of this study was to test whether retroviral insertional mutagenesis contributes to disease onset and whether integration site analysis can identify collaborating genes. The genomic sites of retroviral integration in NUP98-TOP1-induced leukemic mice were analyzed. This screen identified a proviral integration that disrupts expression of the Interferon consensus sequence binding protein (ICSBP) tumor suppressor gene. Intriguingly, an ICSBP deficiency induces a chronic myeloid leukemia-like disease in mice and its reduced expression has been observed in several human leukemias. To ascertain whether an ISCBP deficiency collaborates with NUP98-TOP1 in leukemogenesis, we expressed NUP98-TOP1 in ICSBP(-/-) bone marrow. The in vivo myeloproliferation induced by NUP98-TOP1 was markedly exaggerated with the ICSBP(-/-) deficiency. Moreover, NUP98-TOP1/ICSBP(-/-) mice had a reduced survival compared with NUP98-TOP1/ICSBP(+/+) mice. These results reveal the novel finding of collaboration between the ICSBP tumor suppressor gene and NUP98-TOP1 in leukemogenesis. Moreover they further illustrate the power of retroviral integration site analysis for identifying novel cooperating tumor suppressor genes.

Embryonic stem cell-derived hematopoietic stem cells

Despite two decades of studies documenting the in vitro blood-forming potential of murine embryonic stem cells (ESCs), achieving stable long-term blood engraftment of ESC-derived hematopoietic stem cells in irradiated mice has proven difficult. We have exploited the Cdx-Hox pathway, a genetic program important for blood development, to enhance the differentiation of ESCs along the hematopoietic lineage. Using an embryonic stem cell line engineered with tetracycline-inducible Cdx4, we demonstrate that ectopic Cdx4 expression promotes hematopoietic mesoderm specification, increases hematopoietic progenitor formation, and, together with HoxB4, enhances multilineage hematopoietic engraftment of lethally irradiated adult mice. Clonal analysis of retroviral integration sites confirms a common stem cell origin of lymphoid and myeloid populations in engrafted primary and secondary mice. These data document the cardinal stem cell features of self-renewal and multilineage differentiation of ESC-derived hematopoietic stem cells.

Clonal evidence for the transduction of CD34+ cells with lymphomyeloid differentiation potential and self-renewal capacity in the SCID-X1 gene therapy trial

AbstractImmune function has been restored in 9 of 10 children with X-linked severe combined immunodeficiency by γc gene transfer in CD34+ cells. The distribution of both T-cell receptor (TCR) Vβ family usage and TCR Vβ complementarity-determining region 3 (CDR3) length revealed a broadly diversified T-cell repertoire. Retroviral integration site analysis in T cells demonstrated a high number of distinct insertion sites, indicating polyclonality of genetically corrected cell clones, in all patients. Detection of γc transgene expression on patients' mature myeloid cells has prompted us to investigate the nature of the most immature transduced hematopoietic precursor cells. Insertion sites shared by T and B lymphocytes as well as highly purified granulocytes and monocytes demonstrate the correction of common multipotent progenitor cells. Moreover, our data show that differentiated leukocytes share the same exact insertion sites with CD34+ cells that we obtained 8 months later and that were able to generate long-term culture-initiating cells (LTC-ICs). This finding demonstrates the initial transduction of very primitive multipotent progenitor cells with self-renewal capacity. These results provide a first evidence in the setting of a clinical trial that CD34+ cells maintain both lymphomyeloid potential as well as self-renewal capacity after ex vivo manipulation.

Molecular Characterization of Stem Cell Populations and Specific Clone Tracking of Retrovirally-Marked Chemoprotected Stem Cells before and after Dose-Escalating Chemotherapy.

In vivo selection strategies of genetically modified cells carrying mutant forms of MGMT (i.e. P140K or G156A) have the potential to improve autologous and allogeneic stem cell gene therapy and transplantation. Previously, we have shown efficient in vivo selection and marrow protection in a clinically relevant canine model. Here we describe molecular analysis of stem cell pools before, during, and after aggressive chemotherapy with O6BG and BCNU or temozolomide. LAM-PCR was carried out to identify clones and track those clones over time. This study includes four dogs that received chemotherapy; two dogs received DLA-identical allogeneic transplants (G154/G069) and two dogs received autologous transplants (G197/G179). A control dog received an autologous transplant, but never received chemotherapy (G038) was included. Chemotherapy treated animals received CD34-enriched marrow cells transduced with an RD114-pseudotype retroviral vector encoding P140K and eGFP while the control dog retroviral vector encoded only eGFP. After stable engraftment four dogs were treated with either O6BG and BCNU or temozolomide. Initially, granulocyte marking levels ranged from 10–16% before the dogs received repeated dose-escalating regimens of chemotherapy. After the final round of chemotherapy granulocyte marking was >98% and stabilized at 66–97% with stable increases in all cell lineages analyzed. For LAM-PCR analysis DNA was extracted from peripheral blood leukocytes before chemotherapy (71–95 days post-transplant designated PRE), after 4–6 treatments with chemotherapy (262–354 days post-transplant designated EARLY), and after the last treatment with chemotherapy (476–635 days post-transplant designated LATE). The DNA samples analyzed for G038 corresponded chronologically with that of the treatment animals with samples taken 95, 235, and 601 days post-transplant. Retroviral integration site analysis has shown that the four chemotherapy treated dogs and control dog are polyclonal at all times with no trend towards oligoclonal or monoclonal states. The stem cell pools of G038 are very similar with ~26% of clones identified in every pool after random sampling on bulk DNA. Additionally, overlapping sequences account for ~11% of PRE/EARLY clones, ~16% EARLY/LATE clones, and ~16% PRE/LATE clones. Unique sequences in each group account for about half of the clones analyzed. Conversely, in the dogs treated with chemotherapy, in over 40 clones analyzed no overlap between sequences has been identified. To further characterize the contribution of unique clones specific primers were designed to clones attained from integration sites collected in FACS-sorted populations from G197. When additional FACS-sorted and bulk peripheral blood samples from PRE/EARLY/LATE were probed the specific clone could be identified. This suggests that although the stem cell pool is polyclonal at all time points the clonal contribution of the pool is markedly different. This also suggests that the initial stem cell pool is composed of 100s to 1000s of clones. Retroviral copy number and telomere length of marked cells that have been selected by repeated treatments with chemotherapy is currently ongoing. Our studies suggest that even after aggressive in vivo selection of chemoprotected cells hematopoiesis has remained polyclonal and no side effects or malignancies were observed.

708. Retroviral Vector Integration into the Genome of Rhesus Macaque Long-Term Repopulating Cells

Replication defective retroviruses are very attractive tools for hematopoietic stem cell (HSC) gene therapy largely because of their ability to integrate into genomic DNA. While the risk of insertional mutagenesis has long been recognized, reports of neoplasia consecutive to proto-oncogenes' activation in mice and humans have refocused attention on the safety limitations of these vectors. Mapping studies of retroviral integration sites (RIS) in human cell lines have suggested that retroviral vectors favor integration near transcriptional start sites. Medical applications using these vectors are aimed at HSC, and thus large-scale analysis of RIS in a relevant pre-clinical situation is essential to assess the safety of HSC gene therapy. Here we report the mapping of 423 RIS in a long-term model of autologous retrovirally-transduced HSC transplantation in the non-human primate (Macaca mulatta). Circulating granulocytes and mononuclear cells from 22 rhesus macaques, transplanted with G-CSF + SCF mobilized CD34+ peripheral blood stem cells transduced with an amphotropic Moloney murine leukemia-derived vector containing a neomycin-resistance marker gene, have been analyzed between one and five years after transplantation. Genomic regions adjacent to RIS have been retrieved using linear amplification-mediated PCR followed by shotgun TA cloning, and mapped to the NCBI build 34 of the human genome sequence. Out of the 423 RIS retrieved, 37% landed between the transcriptional start and stop codons of a member of the well-defined RefSeq set of genes, 55% were located in non-coding regions of the genome and 8% were found to be in interspersed repeats and therefore not mappable. Analysis of the targeted genomic regions provides evidence for non-random insertion: 8 independent RIS, recovered from 6 animals, have been mapped within the 2 first introns of the myelodysplasia syndrome 1 gene (MDS1), and seven other genes have 2 independent insertions. Among these 8 genes, it is noteworthy that three are known to be involved in chromosomal rearrangements observed in spontaneous acute human leukemias: the proto-oncogene runt-related transcription factor 1 (RUNX1), the fms-related tyrosine kinase 3 (FLT3) and MDS1. The presence of the fusion sequence between the genomic MDS1 region and the 5'-LTR of the vector has been confirmed using insertion-specific primers for 6 out of the 8 events, and clonal evolution as well as expression of the targeted gene are being analyzed. These observations suggest that retroviral integration in long-term repopulating HSC is highly non-random and is favored at specific loci. Systematic mapping of RIS after HSC gene transfer in the non-human primate will provide critical pre-clinical data necessary for risk assessment of such vectors, and offers also a powerful means for identifying genes involved in the biology of HSC.