Karen Pepper

Long‐Term Cytokine Production from Engineered Primary Human Stromal Cells Influences Human Hematopoiesis in an In Vivo Xenograft Model

Human hematopoiesis can be supported in beige/nude/ XID (bnx) mice by coinjection of human bone marrow stromal cells engineered to secrete human interleukin 3 (HuIL‐3). The major limitation is a total absence of human B cell development in the mice, which could be due to supraphysiological levels of HuIL‐3 in the circulation. In an effort to obtain human B lymphoid, as well as T lymphoid and myeloid cell development in the mice, CD34+ cells were coinjected with human marrow stromal cells engineered to secrete human IL‐2, IL‐7, stem cell factor or FLT3 ligand, ± IL‐3. No single factor other than IL‐3 supported sustained human hematopoiesis in the mice, although cytokines were expressed for four to six months post‐transplantation. Production of both HuIL‐3 and IL‐7 in the mice supported extrathymic development of human T lymphocytes, but no B cells, myeloid cells, or clonogenic progenitors were detected. Human B cells were not produced from CD34+ cells in the bnx mice under any condition tested. Another limitation to the bnx/Hu system is a lack of maturation of human red blood cells, although BFU‐E are maintained. Stromal cells secreting human erythropoietin and IL‐3 were cotransplanted into mice with HuCD34+ cells and an increase in hematocrit from 40%‐45% to 80%‐85% resulted, with production of human and murine red blood cells. Unfortunately, all mice (n = 9) suffered strokes, displayed paralysis and died within three weeks. The bnx/Hu cotransplantation model provides an interesting system in which to study human hematopoietic cell differentiation under the influence of various cytokines.

In vivo biosafety model to assess the risk of adverse events from retroviral and lentiviral vectors.

Serious adverse events in some human gene therapy clinical trials have raised safety concerns when retroviral or lentiviral vectors are used for gene transfer. We evaluated the potential for generating replication-competent retrovirus (RCR) and assessed the risk of occurrence of adverse events in an in vivo system. Human hematopoietic stem and progenitor cells (HSCs) and mesenchymal stem cells (MSCs) transduced with two different Moloney murine leukemia virus (MoMuLV)-based vectors were cotransplanted into a total of 481 immune-deficient mice (that are unable to reject cells that become transformed), and the animals were monitored for 18 months. Animals with any signs of illness were immediately killed, autopsied, and subjected to a range of biosafety studies. There was no detectable evidence of insertional mutagenesis leading to human leukemias or solid tumors in the 18 months during which the animals were studied. In 117 serum samples analyzed by vector rescue assay there was no detectable RCR. An additional 149 mice received HSCs transduced with lentiviral vectors, and were followed for 2-6 months. No vector-associated adverse events were observed, and none of the mice had detectable human immunodeficiency virus (HIV) p24 antigen in their sera. Our in vivo system, therefore, helps to provide an assessment of the risks involved when retroviral or lentiviral vectors are considered for use in clinical gene therapy applications.

Examination of mesenchymal stem cell-mediated RNAi transfer to Huntington's disease affected neuronal cells for reduction of huntingtin.

Huntingtons disease (HD) is a fatal, autosomal dominant neurodegenerative disorder caused by an expanded trinucleotide (CAG) repeat in exon 1 of the huntingtin gene (Htt). This expansion creates a toxic polyglutamine tract in the huntingtin protein (HTT). Currently, there is no treatment for either the progression or prevention of the disease. RNA interference (RNAi) technology has shown promise in transgenic mouse models of HD by reducing expression of mutant HTT and slowing disease progression. The advancement of RNAi therapies to human clinical trials is hampered by problems delivering RNAi to affected neurons in a robust and sustainable manner. Mesenchymal stem cells (MSC) have demonstrated a strong safety profile in both completed and numerous ongoing clinical trials. MSC exhibit a number of innate therapeutic effects, such as immune system modulation, homing to injury, and cytokine release into damaged microenvironments. The ability of MSC to transfer larger molecules and even organelles suggested their potential usefulness as delivery vehicles for therapeutic RNA inhibition. In a series of model systems we have found evidence that MSC can transfer RNAi targeting both reporter genes and mutant huntingtin in neural cell lines. MSC expressing shRNA antisense to GFP were found to decrease expression of GFP in SH-SY5Y cells after co-culture when assayed by flow cytometry. Additionally MSC expressing shRNA antisense to HTT were able to decrease levels of mutant HTT expressed in both U87 and SH-SY5Y target cells when assayed by Western blot and densitometry. These results are encouraging for expanding the therapeutic abilities of both RNAi and MSC for future treatments of Huntingtons disease.

The Moloney murine leukemia virus repressor binding site represses expression in murine and human hematopoietic stem cells

ABSTRACT The Moloney murine leukemia virus (MLV) repressor binding site (RBS) is a major determinant of restricted expression of MLV in undifferentiated mouse embryonic stem (ES) cells and mouse embryonal carcinoma (EC) lines. We show here that the RBS repressed expression when placed outside of its normal MLV genome context in a self-inactivating (SIN) lentiviral vector. In the lentiviral vector genome context, the RBS repressed expression of a modified MLV long terminal repeat (MNDU3) promoter, a simian virus 40 promoter, and three cellular promoters: ubiquitin C, mPGK, and hEF-1a. In addition to repressing expression in undifferentiated ES and EC cell lines, we show that the RBS substantially repressed expression in primary mouse embryonic fibroblasts, primary mouse bone marrow stromal cells, whole mouse bone marrow and its differentiated progeny after bone marrow transplant, and several mouse hematopoietic cell lines. Using an electrophoretic mobility shift assay, we show that binding factor A, the trans-acting factor proposed to convey repression by its interaction with the RBS, is present in the nuclear extracts of all mouse cells we analyzed where expression was repressed by the RBS. In addition, we show that the RBS partially repressed expression in the human hematopoietic cell line DU.528 and primary human CD34+ CD38− hematopoietic cells isolated from umbilical cord blood. These findings suggest that retroviral vectors carrying the RBS are subjected to high rates of repression in murine and human cells and that MLV vectors with primer binding site substitutions that remove the RBS may yield more-effective gene expression.

Human Mesenchymal Stem Cells Genetically Engineered to Overexpress Brain-derived Neurotrophic Factor Improve Outcomes in Huntington’s Disease Mouse Models

Huntingtons disease (HD) is a fatal degenerative autosomal dominant neuropsychiatric disease that causes neuronal death and is characterized by progressive striatal and then widespread brain atrophy. Brain-derived neurotrophic factor (BDNF) is a lead candidate for the treatment of HD, as it has been shown to prevent cell death and to stimulate the growth and migration of new neurons in the brain in transgenic mouse models. BDNF levels are reduced in HD postmortem human brain. Previous studies have shown efficacy of mesenchymal stem/stromal cells (MSC)/BDNF using murine MSCs, and the present study used human MSCs to advance the therapeutic potential of the MSC/BDNF platform for clinical application. Double-blinded studies were performed to examine the effects of intrastriatally transplanted human MSC/BDNF on disease progression in two strains of immune-suppressed HD transgenic mice: YAC128 and R6/2. MSC/BDNF treatment decreased striatal atrophy in YAC128 mice. MSC/BDNF treatment also significantly reduced anxiety as measured in the open-field assay. Both MSC and MSC/BDNF treatments induced a significant increase in neurogenesis-like activity in R6/2 mice. MSC/BDNF treatment also increased the mean lifespan of the R6/2 mice. Our genetically modified MSC/BDNF cells set a precedent for stem cell-based neurotherapeutics and could potentially be modified for other neurodegenerative disorders such as amyotrophic lateral sclerosis, Alzheimers disease, and some forms of Parkinsons disease. These cells provide a platform delivery system for future studies involving corrective gene-editing strategies.Huntingtons disease (HD) is a fatal degenerative autosomal dominant neuropsychiatric disease that causes neuronal death and is characterized by progressive striatal and then widespread brain atrophy. Brain-derived neurotrophic factor (BDNF) is a lead candidate for the treatment of HD, as it has been shown to prevent cell death and to stimulate the growth and migration of new neurons in the brain in transgenic mouse models. BDNF levels are reduced in HD postmortem human brain. Previous studies have shown efficacy of mesenchymal stem/stromal cells (MSC)/BDNF using murine MSCs, and the present study used human MSCs to advance the therapeutic potential of the MSC/BDNF platform for clinical application. Double-blinded studies were performed to examine the effects of intrastriatally transplanted human MSC/BDNF on disease progression in two strains of immune-suppressed HD transgenic mice: YAC128 and R6/2. MSC/BDNF treatment decreased striatal atrophy in YAC128 mice. MSC/BDNF treatment also significantly reduced anxiety as measured in the open-field assay. Both MSC and MSC/BDNF treatments induced a significant increase in neurogenesis-like activity in R6/2 mice. MSC/BDNF treatment also increased the mean lifespan of the R6/2 mice. Our genetically modified MSC/BDNF cells set a precedent for stem cell-based neurotherapeutics and could potentially be modified for other neurodegenerative disorders such as amyotrophic lateral sclerosis, Alzheimers disease, and some forms of Parkinsons disease. These cells provide a platform delivery system for future studies involving corrective gene-editing strategies.

Preclinical evaluation of mesenchymal stem cells overexpressing VEGF to treat critical limb ischemia

Numerous clinical trials are utilizing mesenchymal stem cells (MSC) to treat critical limb ischemia, primarily for their ability to secrete signals that promote revascularization. These cells have demonstrated clinical safety, but their efficacy has been limited, possibly because these paracrine signals are secreted at subtherapeutic levels. In these studies the combination of cell and gene therapy was evaluated by engineering MSC with a lentivirus to overexpress vascular endothelial growth factor (VEGF). To achieve clinical compliance, the number of viral insertions was limited to 1–2 copies/cell and a constitutive promoter with demonstrated clinical safety was used. MSC/VEGF showed statistically significant increases in blood flow restoration as compared with sham controls, and more consistent improvements as compared with nontransduced MSC. Safety of MSC/VEGF was assessed in terms of genomic stability, rule-out tumorigenicity, and absence of edema or hemangiomas in vivo. In terms of retention, injected MSC/VEGF showed a steady decline over time, with a very small fraction of MSC/VEGF remaining for up to 4.5 months. Additional safety studies completed include absence of replication competent lentivirus, sterility tests, and absence of VSV-G viral envelope coding plasmid. These preclinical studies are directed toward a planned phase 1 clinical trial to treat critical limb ischemia.

Human Myoblast and Mesenchymal Stem Cell Interactions Visualized by Videomicroscopy.

Muscle-derived progenitor cell (myoblast) therapy has promise for the treatment of denervated, weakened, and fibrotic muscle. The best methods for injecting myoblasts to promote fusion and retention have yet to be determined, however. Mesenchymal stem/stromal cells have also been reported to have beneficial effects in restoring damaged tissue, through increasing vascularization and reducing inflammation. The interactions between human primary skeletal myoblasts and bone marrow-derived mesenchymal stem/stromal cells were examined using time-lapse images put into video format. Of interest, there is a high degree of cell-to-cell interaction with microparticles transferring between both cell types, and formation of nanotubules to bridge cytoplasmic contents between the two types of cell. This model provides an in vitro platform for examining mechanisms for cell-to-cell interaction preceding myoblast fusion.

462. Busulfan Dose Escalation to Increase Gene Marking of Hematopoietic Stem Cells by Lentiviral Vectors in Infant Rhesus Monkeys

Top of pageAbstract In clinical allogeneic bone marrow transplantation (BMT), complete myeloablation with high-dose busulfan (16 mg/kg) is often used to |[ldquo]|make space|[rdquo]| for the graft in the recipient BM compartment. As this treatment has significant toxicity, we sought to establish a low-dose busulfan protocol for partial myeloablation in the context of a gene-modified autologous BMT. Also, for infants and young children, calculation of dosage based on body surface area (mg/m2) has been reported give more consistent circulating busulfan levels than dosages based on mass (kg). In this study we sought to identify an optimal busulfan dose that could result in efficient long-term gene marking with minimal toxicities. We performed a lentiviral gene-marking study in infant rhesus macaques using escalating doses of busulfan. BM (|[sim]|10 ml/kg) was collected for immunoselection, and followed by a single 2 hour i.v. infusion of busulfan. Monkeys were transplanted using busulfan at 0, 40, 80, 120, and 160 mg/m2. Peripheral blood (PB) samples were collected for pharmacokinetic analysis up to 4 hours post-infusion, and the area under the curve (AUC) was determined. Isolated CD34+ cells were cultured for 24 hours in X-Vivo 15 with 100 ng/ml each of SCF, Flt3-L, and TPO. CD34+ cells were then transduced overnight with 4|[times]|10e7 infectious particles/ml of Simian Immunodeficiency Virus (SIV)-derived lentiviral vector pseudotyped with VSV-G on fibronectin-coated plates and using 4 |[mu]|g/ml protamine sulfate. The SIV vector contains a neomycin gene with a mutation in the start codon that abolishes its expression, and can therefore serve as a non-expressed marker gene. Transduced cells were washed and reinfused i.v 48 hours after administration of busulfan. Animals were monitored for myelosuppression, toxicity, and immune function. Increasing dosages of busulfan resulted in increased AUC. Some variability in AUC at each dose level was observed, suggesting inter-individual variations in busulfan absorption and clearance. At busulfan doses of 120 and 160 mg/m2, neutrophil and platelet counts transiently declined and were dose-dependent. No lymphopenia was observed, consistent with busulfan being myeloablative, but not immunosuppressive. Blood chemistries and behavior were normal in all animals, and no abnormalities were observed. PB and BM samples collected monthly were analyzed by real-time PCR to quantify gene marking. Gene marking levels have been fairly constant over the first 4 months post-transplant, ranging from undetectable in animals receiving no busulfan up to 0.1 % gene-containing cells in animals with the highest busulfan AUC. Together, these preliminary data suggest that (1) increased gene marking can be achieved by escalating busulfan doses, (2) busulfan is safe at the sub-myeloablative doses studied, and (3) while the anticipated myelosuppressive effects of busulfan were observed, there has been no evidence of adverse findings, to date.

1102. Stable, Non-Viral Gene Transfer to Human CD34+ Hematopoietic Progenitor Cells Using the Sleeping Beauty Transposon

Primary hematopoietic stem cells (HSC), capable of repopulating the entire hematopoietic and immune system with the full complement of cell types, are an attractive target for gene therapy strategies to treat diseases of blood cells. Gene modification of a patients HSC could be used for autologous transplantation to treat inherited genetic disorders, including Severe Combined Immune Deficiency (SCID) and other immune deficiencies. Non-viral strategies have become available for gene transfer into cells that result in integration of therapeutic gene cassettes. For example, Sleeping Beauty (SB) transposes directly from one DNA locus to another through the activity of the transposase enzyme recognizing and performing excision and integration of the DNA sequences between specific inverted repeat (IR) sequences of the transposon. Transposase-mediated integration of the IR-flanked expression cassette into chromosomes provides the basis for long-term transgene expression. Here we used the transposon-based gene delivery strategy composed of a two plasmid system: a plasmid containing the SB transposase gene (for transient production of the transposase), and a second plasmid containing an expression cassette with a reporter gene flanked by the SB IR (inverted repeat) sequences. For this study, we evaluated the potential use of the SB transposon system to deliver genes in a stable manner to human hematopoietic cells by electroporation using the Amaxa nucleoporator. One plasmid carrying an enhanced Green Fluorescent Protein (eGFP) reporter cassette (to facilitate FACS analysis of resultant cells) flanked by the SB IR sequences for permanent integration into the target cell chromosomes was co-nucleoporated with a second plasmid carrying the SB transposase expression cassette. We first used the K562 human erythro-leukemia cell line to optimize several parameters of gene delivery, achieving high levels (50-80%) of stable gene integration with optimal amounts and ratios of the two plasmids. Southern analysis of K562 clonal populations showed that 1 to 6 copies of the transposon can be detected. We then nucleoporated the SB-based system into human CD34+ progenitor cells isolated from umbilical cord blood and achieved initial gene transfer to these primary cells at moderate levels (10-30%) that led to stable expression of the reporter gene (1-4%). Results from these studies demonstrate the potential for using the SB transposon system for gene therapy using HSC.

408. Developing Lentiviral Vectors for Specific Gene Expression in CD4+ T Cells

In certain applications of gene therapy, regulated (rather than constitutive) gene expression will be crucial for proper function or optimal effect of the delivered gene. Therefore, an important goal of gene therapy is to be able to deliver genes so that they express in a pattern that recapitulates the expression of an endogenous cellular gene. Additionally, regulating transgene expression will be an important component of efforts for reducing risks of adverse advents associated with gene therapy with integrating vectors.