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Lentiviral vectors are increasingly utilized in cell and gene therapy applications because they efficiently transduce target cells such as hematopoietic stem cells and T cells. Large-scale production of current Good Manufacturing Practices-grade lentiviral vectors is limited because of the adherent, serum-dependent nature of HEK293T cells used in the manufacturing process. To optimize large-scale clinical-grade lentiviral vector production, we developed an improved production scheme by adapting HEK293T cells to grow in suspension using commercially available and chemically defined serum-free media. Lentiviral vectors with titers equivalent to those of HEK293T cells were produced from SJ293TS cells using optimized transfection conditions that reduced the required amount of plasmid DNA by 50%. Furthermore, purification of SJ293TS-derived lentiviral vectors at 1 L yielded a recovery of 55% ± 14% (n = 138) of transducing units in the starting material, more than a 2-fold increase over historical yields from adherent HEK293T serum-dependent lentiviral vector preparations. SJ293TS cells were stable to produce lentiviral vectors over 4 months of continuous culture. SJ293TS-derived lentiviral vectors efficiently transduced primary hematopoietic stem cells and T cells from healthy donors. Overall, our SJ293TS cell line enables high-titer vector production in serum-free conditions while reducing the amount of input DNA required, resulting in a highly efficient manufacturing option.

Hematopoietic stem cell gene therapy for hemoglobin disorders, including sickle cell disease, requires high-efficiency lentiviral gene transfer and robust therapeutic globin expression in erythroid cells. Erythropoietin is a key cytokine for erythroid proliferation and differentiation (erythropoiesis), and truncated human erythropoietin receptors (thEpoR) have been reported in familial polycythemia. We reasoned that coexpression of thEpoR could enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous nonhuman primate transplantation models. We generated thEpoR by deleting 40 amino acids from the carboxyl terminus, allowing for erythropoietin-dependent enhanced erythropoiesis of gene-modified cells. We then designed lentiviral vectors encoding both thEpoR and B cell lymphoma/leukemia 11A (BCL11A)–targeting microRNA-adapted short hairpin RNA (shmiR BCL11A) driven by an erythroid-specific promoter. thEpoR expression enhanced erythropoiesis among gene-modified cells in vitro. We then transplanted lentiviral vector gene-modified CD34+ cells with erythroid-specific expression of both thEpoR and shmiR BCL11A and compared to cells modified with shmiR BCL11A only. We found that thEpoR enhanced shmiR BCL11A–based fetal hemoglobin (HbF) induction in both xenograft mice and rhesus macaques, whereas HbF induction with shmiR BCL11A only was robust, yet transient. thEpoR/shmiR BCL11A coexpression allowed for sustained HbF induction at 20 to 25% in rhesus macaques for 4 to 8 months. In summary, we developed erythroid-specific thEpoR/shmiR BCL11A–expressing vectors, enhancing HbF induction in xenograft mice and rhesus macaques. The sustained HbF induction achieved by addition of thEpoR and shmiR BCL11A may represent a viable gene therapy strategy for hemoglobin disorders.

Wiskott-Aldrich syndrome (WAS) is a life-threatening immunodeficiency caused by mutations within the WAS gene. Viral gene therapy to restore WAS protein (WASp) expression in hematopoietic cells of patients with WAS has the potential to improve outcomes relative to the current standard of care, allogeneic bone marrow transplantation. However, the development of viral vectors that are both safe and effective has been problematic. While use of viral transcriptional promoters may increase the risk of insertional mutagenesis, cellular promoters may not achieve WASp expression levels necessary for optimal therapeutic effect. Here we evaluate a self-inactivating (SIN) lentiviral vector combining a chromatin insulator upstream of a viral MND (MPSV LTR, NCR deleted, dl587 PBS) promoter driving WASp expression. Used as a gene therapeutic in Was−/− mice, this vector resulted in stable WASp+ cells in all hematopoietic lineages and rescue of T and B cell defects with a low number of viral integrations per cell, without evidence of insertional mutagenesis in serial bone marrow transplants. In a gene transfer experiment in non-human primates, the insulated MND promoter (driving GFP expression) demonstrated long-term polyclonal engraftment of GFP+ cells. These observations demonstrate that the insulated MND promoter safely and efficiently reconstitutes clinically effective WASp expression and should be considered for future WAS therapy.

Autologous gene therapy using lentiviral vectors (LVs) holds promise for treating monogenetic blood diseases. However, clinical applications can be limited by suboptimal hematopoietic stem cell (HSC) transduction and insufficient quantities of available vector. We recently reported gene therapy for X-linked severe combined immunodeficiency using a protocol in which patient CD34+ cells were incubated with two successive transductions. Here we describe an improved protocol for LV delivery to CD34+ cells that simplifies product manipulation, reduces vector consumption, and achieves greater vector copy number (VCN) of repopulating HSCs in mouse xenotransplantation assays. Notable findings include the following: (1) the VCN of CD34+ cells measured shortly after transduction did not always correlate with the VCN of repopulating HSCs after xenotransplantation; (2) single-step transduction at higher CD34+ cell concentrations (2–4 × 106/ml) conserved LV without compromising HSC VCN; (3) poloxamer F108 (LentiBOOST) increased HSC VCN by two- to threefold (average from three donors); (4) although LentiBOOST + prostaglandin E2 combination further increased VCN in vitro, the VCN observed in vivo were similar to LentiBOOST alone; (5) cyclosporine H increased the HSC VCN to a similar or greater extent with LentiBOOST in vivo. Our findings delineate an improved protocol to increase the VCN of HSCs after CD34+ cell transduction with clinically relevant LVs.

Humanized mouse models have been developed to study human hematopoiesis and therapeutic application of hematopoietic stem cell transplantation. To evaluate the safety and efficacy of lentiviral vectors for gene therapy, human CD34+ cells have been transduced with lentiviral vectors and transplanted into the humanized mice. Recipient mice are monitored over time and sacrificed for bone marrow analyses with regard to human cell engraftment, lineage distribution, and vector transduction. This chapter details the procedure for lentiviral transduction and transplantation of human hematopoietic stem/progenitor cells into humanized mice to study inherited human hematological disorders.

Hematopoietic stem cell gene therapy for hemoglobin disorders, such as sickle cell disease, requires high-level gene marking and robust therapeutic globin expression in erythroid cells (>20% of γ- or β-globin production) for widespread successful clinical application. We previously demonstrated that lentiviral transduction of a truncated human erythropoietin receptor (thEpoR) gene allows for erythropoietin-dependent selective proliferation of gene-modified human erythroid cells during in vitro differentiation (ASH 2017). In this study, we sought to evaluate whether thEpoR can enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous non-human primate transplantation models. To investigate this hypothesis, we designed lentiviral vectors encoding both thEpoR and BCL11A-targeting micro RNA-adapted short hairpin RNA (shmiBCL11A), driven off an erythroid specific ankyrin 1 (ANK1) promoter. Both selective proliferation and high-level fetal hemoglobin (HbF) induction were observed in in vitro erythroid differentiation cultures using transduced human CD34+ cells. Healthy donor CD34+ cells were transduced with shmiBCL11A vector, thEpoR-shmiBCL11A vector, and GFP vector (control). Transduced cells were transplanted into immunodeficient NBSGW mice. Five months post-transplant, xenograft bone marrow cells were evaluated for human cell engraftment (human CD45+) and vector copy number (VCN) in both human CD34+ progenitor cells and glycophorin A+ (GPA+) erythroid cells. HbF production was also measured in GPA+ erythroid cells by reverse phase HPLC. We observed efficient transduction in transduced CD34+ cells in vitro (VCN 2.1-5.1) and similar human cell engraftment among all groups (84-89%). The VCN with thEpoR-shmiBCL11A transduction was 3-fold higher in human erythroid cells when compared to CD34+ cells (p We then performed autologous rhesus CD34+ cell transplantation using either shmiBCL11A vector (142562 and RA0706, n=2, compared to a GPA promoter-derived shmiBCL11A vector) or thEpoR-shmiBCL11A vector (ZL50 and ZM24, n=2, compared to a Venus-encoding vector). Transduced CD34+ cells were transplanted into autologous rhesus macaques following 2x5Gy total body irradiation. Efficient transduction was observed in CD34+ cells in vitro among all 4 macaques (VCN 3.8-8.7) using a high-density culture protocol (Uchida N, Mol Ther Methods Clin Dev. 2019). In shmiBCL11A transduction animals, engraftment of gene-modified cells (VCN 0.2-1.0) and robust HbF induction (14-16%) were observed 1 month post-transplant. However, VCN and HbF levels were reduced down to VCN ~0.1 and HbF ~0.4% in both animals 6 months post-transplant. In contrast, a thEpoR-shmiBCL11A transduction animal (ZL50) resulted in engraftment of gene-modified cells (VCN 0.8-1.0) and robust HbF induction (~18%) 1 month post-transplant, with both gene marking and HbF levels remaining high at VCN 0.6-0.7 and HbF ~15% 4 months post-transplant. These data suggest that shmiBCL11A transduction results in transient HbF induction in gene-modified erythroid cells, while thEpoR-based selective advantage allows for sustained HbF induction with shmiBCL11A. In summary, we developed erythroid-specific thEpoR-shmiBCL11A expressing vectors, enhancing HbF induction in gene-modified erythroid cells in xenograft mice and rhesus macaques. While further in vivo studies are desirable, the use of thEpoR appears to provide a selective advantage for gene-modified erythroid cells in gene therapy strategies for hemoglobin disorders. Disclosures No relevant conflicts of interest to declare.

Gene therapy for the treatment of Wiskott-Aldrich syndrome (WAS) presents an alternative to the current use of allogeneic bone marrow transplantation. We describe the development of a self-inactivating lentiviral vector containing chromatin insulators for treatment of WAS and compare a gammaretroviral (MND), human cellular (EF1α), and the human WASp gene promoter for expression patterns in vivo during murine hematopoiesis using the green fluorescent protein (GFP) marker. Compared with the EF1α and the WASp promoters, expression from the MND promoter in mouse transplant recipients was much higher in all lineages examined. Importantly, there was sustained expression in the platelets of secondary recipient animals, necessary to correct the thrombocytopenia defect in WAS patients. Analysis of WAS protein expression in transduced human EBV-immortalized B-cells and transduced patient peripheral blood mononuclear cells also demonstrated stronger expression per copy from the MND promoter compared with the other promoters. In addition, when analyzed in an LM02 activation assay, the addition of an insulator to MND-promoter-containing constructs reduced transactivation of the LM02 gene. We propose a clinical trial design in which cytokine-mobilized, autologous, transduced CD34(+) cells are administered after myelosuppression.

Lentiviral vectors are useful for transducing primitive hematopoietic cells. We examined four envelope proteins for their ability to mediate lentiviral transduction of mobilized human CD34(+) peripheral blood cells. Lentiviral particles encoding green fluorescent protein (GFP) were pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein or the feline leukemia virus type C envelope protein. Because the relative amount of genome RNA per ml was similar for each pseudotype, we transduced CD34(+) cells with a fixed volume of each vector preparation. Following an overnight transduction, CD34(+) cells were transplanted into immunodeficient mice which were sacrificed 12 weeks later. The average percentages of engrafted human CD45(+) cells in total bone marrow were comparable to that of the control, mock-transduced group (37-45%). Lenti-particles pseudotyped with the VSV-G envelope protein transduced engrafting cells two- to tenfold better than particles pseudotyped with any of the gamma-retroviral envelope proteins. There was no correlation between receptor mRNA levels for the gamma-retroviral vectors and transduction efficiency of primitive hematopoietic cells. These results support the use of the VSV-G envelope protein for the development of lentiviral producer cell lines for manufacture of clinical-grade vector.

Chromosome Conformation Capture (3C) technology was used to identify physical interactions between the proximal Wiskott-Aldrich Syndrome protein (WASp) promoter and its distant DNA segments in Jurkat-T cells. We found that two hematopoietic specific DNase I hypersensitive (DHS) sites (proximal DHS-A, and distal DHS-B) which had high interaction frequencies with the proximal WASp promoter indicating potential regulatory activity for these DHS sites. Subsequently, we cloned several DNA fragments around the proximal DHS-A site into a luciferase reporter vector. Interestingly, no fragments showed enhancer activity, but two fragments exhibited strong silencing activity in Jurkat-T cells. After aligning the chromatin state profiling for hematopoietic and nonhematopoietic cells using the human genome browser (UCSC), we found a 5 kb putative hematopoietic specific enhancer region located 250 kb downstream of the WAS gene. This putative enhancer region contains two hematopoietic cell specific DHS sites. Subsequently, the hematopoietic specific DHS sites enhanced luciferase expression from the proximal WASp promoter in all hematopoietic cells we tested. Finally, using a lentiviral vector stable expression system, the hematopoietic specific-enhancer(s) increased GFP reporter gene expression in hematopoietic cells, and increased WASp gene expression in WASp deficient cells. This enhancer may have the potential to be used in gene therapy for hematological diseases.

Lentiviral vectors efficiently transduce quiescent stem cells and are being evaluated for gene therapy of blood dis-orders. The risk of genotoxicity as a result of insertional mutagenesis is an important safety consideration. The hy-persensitive site 4 insulator from the chicken beta-globin locus (cHS4) possesses chromatin bar-rier and enhancer-blocking functions. A control lentiviral vector encoding green fluorescent protein was compared with a vector in which the cHS4 insulator element flanked the green fluorescent protein expression cassette in single cell isolates of transduced human T cells (Jurkat) after 9 days in culture. The insulator had minimal effect on mean fluorescent intensity and only modestly reduced the variability of green fluorescent protein expression among indi-vidual single cell isolates. Most unique integration sites were within genes, but the insulator-containing vector had a moderate predilection to integrate near the transcriptional start site compared with the control vector. Clonal domi-nance developed in cultures of cells containing the integrated vector genomes, as reflected by the recovery of mul-tiple single cell isolates containing the same integration site. We infer that certain integrations conferred a prolifera-tive or survival advantage by affecting gene expression through insertional mutagenesis, leading to this clonal dominance. This effect was diminished by including the insulator element in the vector genome.

Cholesterol-rich plasma membrane microdomains are important for entry of many viruses, including retroviruses. Depletion of cholesterol with 2-hydroxypropyl-beta-cyclodextrin inhibits entry of human T cell leukemia virus type I (HTLV-1) and HTLV-I envelope pseudotyped lentivirus particles. Using a soluble fusion protein of the HTLV-I surface envelope protein with the immunoglobulin Fc domain, the HTLV-I receptor was found to colocalize with a raft-associated marker and to cluster in specific plasma membrane microdomains. Depletion of cholesterol did not alter receptor binding activity, suggesting a requirement for cholesterol in a postbinding virus entry step.

The human T-cell leukemia virus type 1 (HTLV-1) envelope protein is required for virus spread. This study further characterizes the role of the envelope protein in HTLV-1 immortalization. Viruses with single amino acid substitutions within the SU protein at residue 75, 81, 95, 101, 105, or 195 or with a C-terminal cytoplasmic domain truncation (CT), as well as an envelope-null (EN) virus, were generated within an infectious molecular clone, ACH. Transfection of 293T cells resulted in the release of similar amounts of virus particles from all of the mutants as determined by p19 enzyme-linked immunosorbent assay and immunoblot analysis of Gag in cell lysates and supernatants. The virus particles from all mutants except ACH-101, ACH-CT, and ACH-EN were infectious for B5 macaque cells in cell-free and cell-to-cell transmission assays and were capable of immortalizing transfected CD4+lymphocytes. These results indicate that HTLV-1 spread is required for immortalization.

Lentiviral vector gene therapy for hematopoietic disorders caused by single gene mutation or deficiency has demonstrated success in clinical trials. However, efficient gene delivery is still challenging, requiring high multiplicity of infection (MOI) to achieve average vector copy numbers of at least 1 per cell. Conditions utilizing high MOI result in improved transduction rates and adequate copy number but these benefits can coincide with potential insertional mutagenesis. Attempting to achieve optimal gene transfer at reduced MOI, we directly compared self-inactivating (SIN) lentiviral vector particles encoding eGFP under control of a murine stem cell virus (MSCV) promoter/enhancer pseudotyped with envelope glycoproteins for BaEV or vesicular stomatitis virus (VSV). BaEV proteins were either full-length (BaWT) or lacked the terminal R-peptide (BaR-less), a version previously reported to improve infectious titer (TU, transducing units per mL) using transient production procedures. Using a standardized four-plasmid transient production protocol, cells transfected with R-less expression plasmids produced syncitia; an issue that resolved using BaWT. Titers were significantly higher for virus packaged with BaWT compared to R-less (BaR-less: 3.6 × 105 ± 6.6 × 104 N=12; BaWT 2.4 × 106 ± 4.5 × 105; n=10; p

The Sindbis virus minimal subgenomic mRNA promoter (spanning positions −19 to +5 relative to the subgenomic mRNA start site) is approximately three- to sixfold less active than the fully active −98 to +14 promoter region. We identified two elements flanking the −19 to +5 region which increase its transcription to levels comparable to the −98 to +14 region. These elements span positions −40 to −20 and +6 to +14 and act synergistically to enhance transcription. Nine different virus libraries were constructed containing blocks of five randomized nucleotides at various positions in the −40 to +14 region. On passaging these libraries in mosquito cells, a small subset of the viruses came to dominate the population. Sequence analysis at the population level and for individual clones revealed that in general, wild-type bases were preferred for positions −15 to +5 of the minimal promoter. Base mutagenesis experiments indicated that the selection of wild-type bases in this region was primarily due to requirements for subgenomic mRNA transcription. Outside of the minimal promoter, the −35 to −29 region contained four positions which also preferred wildtype bases. However, the remaining positions generally preferred non-wild-type bases. On passaging of the virus libraries on hamster cells, the −15 to +5 region again preferred the wild-type base but most of the remaining positions exhibited almost no base preference. The promoter thus consists of an essential central region from −15 to +5 and discrete flanking sites that render it fully active, depending on the host environment.

Sindbis virus (SIN), the type alphavirus, has been studied extensively to identify the viral cis-acting sequences and proteins involved in RNA transcription and replication. However, very little is known about how these processes are coordinated. For example, synthesis of the genomic RNA and the subgenomic mRNA depends on the minus strand. Do these activities occur independently on different templates, or can replication and transcription take place simultaneously on the same template? We describe the appearance of a SIN-specific, plus-sense RNA that is intermediate in size between the genomic and subgenomic RNA species. This RNA, designated RNA II, is observed in a number of different cell lines, both early and late in infection. The number of RNA II species, their sizes, and their abundances are influenced by the subgenomic promoter. We have mapped the 3' end of RNA II to a site within the subgenomic promoter, four nucleotides before the initiation site of the subgenomic mRNA. Our results indicate that the appearance of RNA II is correlated with subgenomic mRNA transcription, such that strong or active promoters tend to increase the abundance of RNA II, relative to weak or less active promoters. RNA II is most abundantly detected with the full promoter and is at much lower abundance with the minimal promoter. The possible origins of RNA II are discussed.

Abstract 164FN2 Somatic gene therapy for X-linked severe combined immunodeficiency (X-SCID) using a MLV-based gammaretroviral vector expressing the IL-2 receptor gamma chain (MFG-γc) resulted in excellent immunologic reconstitution but also in insertional oncogenesis. In 5/20 treated children, T cell leukemia developed, with insertional activation of LMO2 proto-oncogene in 4 of the 5. We reasoned that replacing the virus-derived promoter and enhancer elements with a weaker cellular promoter would result in improved safety yet retain efficacy. We therefore generated the pSRS11.EFS.IL2RG.pre* self-inactivating (SIN) gammaretroviral vector in which expression of γc is controlled by an intronless EF1-α promoter, in a MLV vector devoid of the LTR U3 (enhancer/promoter) region. In preclinical studies we determined ‘relative safety' using several surrogate assays. In a reporter assay, the pSRS11.EFS.IL2RG.pre* vector when inserted into the oncogenic LMO2 locus induced LMO2 expression 6–90-fold less than the parent MFG vector (MFG-γc). pSRS11.EFS.IL2RG.pre* had lower activity in a murine in vitro immortalization assay (0.2 clones per 10e5 cells, fitness score 0.00007), compared to MFG-γc (0.54 clones per 10e5 cells, fitness score 0.00025). Peripheral blood and bone marrow from C57BL6 mice transplanted with murine bone marrow transduced with pSRS11.EFS.IL2RG.pre* and followed in primary recipients over 4 months and in secondary recipients over 1 year in vivo showed no evidence of vector associated leukemias. Deep sequencing demonstrated 8 of 3621 insertions into the MDS-associated gene Evi1 in mice transplanted with MFG-γc-transduced cells while there were none (0 of 2690 insertions into Evi1) in mice transplanted with pSRS11.EFS.IL2RG.pre* vector transduced cells (P=0.025). In preclinical efficacy studies, circulating T and B lymphocytes were detectable in the peripheral blood of 7/7 γc-deficient mice transplanted with pSRS11.EFS.IL2RG.pre* transduced cells while 4/4 mice repopulated with SFFV-eGFP transduced cells remained alymphoid. Experimental animals were sacrificed approximately five months post-transplant for analysis of immune reconstitution. Flow cytometric analysis of the spleens and bone marrow revealed restoration of mature B220+IgM+ B cells and NK cell populations in all mice transplanted with pSRS11.EFS.IL2RG.pre* transduced cells. CD4+ and CD8+ T cells were also detected in both tissues and in thymi recovered from transplanted animals. T cells in these mice proliferated in response to mitogenic stimuli. Immunoglobulin subclasses IgG1 and IgG2a detected in the plasma from pSRS11.EFS.IL2RG.pre* reconstituted mice also indicated restored B cell function in these animals. Human preclinical studies also supported the correction of XSCID cellular defects using this vector. Based on these data, a multi-institutional phase I/II trial was initiated with the pSRS11.EFS.IL2RG.pre* vector using an identical clinical protocol as in the previous X-SCID trials, to determine efficacy and safety compared with the MFG-γc vector. The first patient was treated in December 2010. Six months post-gene therapy, he has attained CD3 T cell count of >800, normal proliferation to mitogens, and normal NK cell numbers. He has cleared medically-resistant oral ulcers, and a rotavirus infection acquired post-gene therapy. Nearly all of the circulating T cells (86%) and 41% of his NK cells express γc, albeit at modestly lower density than normal, as expected. However, the early kinetics of T cell reconstitution was comparable to several subjects treated with MFG-γc. These data suggest that the improved safety profile demonstrated with numerous surrogate preclinical studies is associated with efficacious transgene expression and functional immune recovery in the initial human patient treated. Disclosures: Off Label Use: CliniMACS for selection of CD34+ hematopoietic cells. Baum:Patent office: Patents & Royalties.

Retroviral vector-mediated activation of the LMO2 oncogene led to the onset of leukemia in two X-SCID patients who underwent gene therapy (Science 302: 415, 2003). This has necessitated the development of safer vectors for use in a therapeutic setting. Many pre-clinical studies have now incorporated the use of insulator elements that have boundary and/or enhancer blocking functions to isolate the transcriptional unit within the genome. The chicken beta-globin 5' DNase I hypersensitive site 4 (5'HS4) insulator possesses both of these properties and has been used to overcome positional variegation effects on transgene expression. Characterizing this aspect of insulator function is essential, but it is also of interest to know if there is any differential effect on the pattern of retroviral integration. We are developing lentiviral vectors for gene therapy of Wiskott-Aldrich syndrome (WAS) because of their greater efficiency at transducing repopulating stem cells (Blood 103:4062, 2004) compared to oncoretroviral vectors. WAS is a rare X-linked recessive disorder caused by mutations in the WASP gene which affect cells of the hematopoietic lineage. We have previously demonstrated correction of the T-cell proliferation defect, partial correction of the cytokine secretion defect and improvement in the impaired secondary response to influenza virus in Wasp- mice using MSCV onco-retroviral vectors (Blood 102:3108, 2003). Due to the modest selective advantage of gene-corrected lymphocytes and the variability in the level of gene expression, we are now exploring the use of lentiviral vectors. The efficiency of stem cell-targeted gene transfer achieved with lentiviral vectors and the consistency of gene expression are crucial factors that will determine the success of any therapeutic application for the treatment of WAS. Towards this goal, we are evaluating various vector designs in mice using GFP as a marker gene. To examine the effect of the 5'HS4 insulator within the context of our lentiviral backbone, we have generated advanced third generation vectors containing a modified MSCV promoter (Mp) driving GFP with or without the 5'HS4 insulator. Several single- and multiple-copy T-cell (Jurkat) clones have been isolated for each construct and their expression characterized by flow cytometry. Inclusion of the 5'HS4 insulator did not significantly affect the average mean fluorescence intensity (MFI = 370|[plusmn]|221 versus 329|[plusmn]|120[5'HS4+]), but did reduce the variability of expression amongst individual clones as indicated by a decrease in the co-efficient of variation from 57|[plusmn]|26 to 40|[plusmn]|3. To determine if this insulator has an effect on the position or frequency of insertion, the specific integration sites in clones containing the proviral genomes are being characterized by ligation-mediated (LM)-PCR.

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive disorder caused by mutations in the WASP gene. WASP is an effector protein in the actin polymerization pathway. Characterized by immunodeficiency, micro-thrombocytopenia and eczema, untreated WAS patients may also suffer hemorrhages, recurring infections and potential malignancies. WAS can be cured by bone marrow (BM) transplant but because many patients lack a suitable donor, stem cell-targeted gene transfer is being developed as an alternative therapeutic approach. We have demonstrated correction of the T-cell proliferation defect in Wasp− mice using MSCV oncoretroviral vectors (Blood102:3108, 2003). However, our competitive repopulation studies in mice with both wild-type (WT) and gene-corrected Wasp− BM, demonstrated only a modest selective advantage for gene modified lymphocytes. Correction of the lymphocytopenia was observed only in animals having high proportions of transduced cells. In addition, variability in the level of gene expression among gene-corrected cells was associated with only partial correction of the T-cell cytokine secretory defects. Thus, the efficiency of stem cell-targeted gene transfer as well as the level and consistency of gene expression are likely to be key factors that determine success in any clinical application of gene transfer for the treatment of WAS. In attempting to improve vector design, we have used lentiviral vectors because of their greater efficiency in transducing repopulating stem cells as we recently demonstrated in a non-human primate model (Blood103:4062, 2004). The WASP gene is regulated by two promoter regions. The proximal promoter lies immediately adjacent to the translation start site with the distal promoter found 6 kb upstream, followed by an alternate first exon. We have developed a series of third generation, self-inactivating lentiviral vectors containing the MSCV, proximal or distal WASP promoters driving GFP in the reverse transcriptional orientation. The WASP promoters were active in lymphocytes but not HeLa cells in vitro. However, expression was low in lymphocytes and granulocytes in mice transplanted with genetically modified stem cells. In an effort to abrogate any position-effect variegation and enhance expression, we generated a new series of vectors with the transcriptional unit in the forward orientation that also contained the woodchuck post-transcriptional regulatory element, the chicken beta-globin 5′ DNase I hypersensitive site 4 (I) and human beta-interferon scaffold attachment region (S) insulator elements. Either GFP or murine Wasp cDNA were included for expression analysis. Despite the complexity of the SI-containing vectors, titers of concentrated preparations ranged from 9x106 TU/ml to 5x107 TU/ml and enabled transduction of both cell lines and murine hematopoietic stem cells. Low-level GFP expression from the proximal promoter was detected in HeLa cells with higher expression found in lymphocytes (NALM6 and Jurkat cells). Expression of GFP under the control of the proximal WASP promoter was detected in vivo in multiple hematopoietic lineages in mice transplanted with transduced stem cells. Future efforts will focus on further characterization and optimization of vector design with the goal of achieving consistent, high level expression.