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Introduction Platelets may comprise an ideal vehicle for delivering FVIII in hemophilia A (HemA) as FVIII stored in platelet α-granules is protected from neutralization by inhibitory antibodies and, during bleeding, activated platelets locally excrete their contents to promote clot formation. In order to avoid specific challenges posed by ex vivo gene delivery including, in particular, the requirement to pre-condition the subject, we evaluated intraosseous (IO) infusion of self-inactivating lentiviral vectors (LV) for in situ gene transfer into bone marrow cells. In previous studies, we confirmed that hematopoietic stem cells (HSCs) can be efficiently transduced to express GFP after IO administration of LV driven by a MND promoter (M-GFP-LV). Methods In the current study, we aimed at limiting transgene expression to the megakaryocyte lineages using IO delivery of 20 µL LV containing either GFP (G-GFP-LV) or a B-domain variant human FVIII (G-F8-LV) gene under the control of megakaryocyte strictly specific promoter glycoprotein 1bα (Gp1bα). Results In M-GFP-LV treated control mice, GFP was detected in 6.4% of HSCs, 3.4% of B220+, and 9.0% of CD11c+ bone marrow cells on day 29. In contrast, in G-GFP-LV (6.0E+08 TU/mL) treated mice, GFP was undetectable in bone marrow HSCs, B220+, CD11c+ or CD11b+ cells. GFP expression level in platelets of G-GFP-LV treated mice was ten folds of that in M-GFP-LV treated mice (0.1% vs 0.01%). It indicated that in platelets, the activity of Gp1bα was stronger than that of MND. More importantly, GFP expression levels were stable over 100 days, suggesting that platelets containing the transgene products did not elicit transgene-specific immune responses. Next, we treated HemA mice with G-F8-LV (6.0 E+07 TU/mL). There was no detectable hFVIII expression in bone marrow HSCs on day 8 or in blood cells (CD3ε+, B220+, CD11c+ or CD11b+) on day 35. However, up to 3% platelets express hFVIII on day 91. These results suggested that HSCs in HemA mice were successfully transduced by G-F8-LV after IO infusion, and in the long term, FVIII was synthesized in megakaryocytes and stored in platelet α-granules. In treated mice, the average percentage of platelets expressing hFVIII was stable at 1-2% from day 27 to day 160. The average FVIII antigen level in platelets on day 112 was 1 mU per 1 × 108 platelets, which was comparable with platelet FVIII in transgenic and ex vivo gene therapy treated mice. We also evaluated LV-treated HemA animals for phenotypic correction of bleeding diathesis by tail clip assay. The blood loss was 41% (n=7), 48% (n=5) and 33% (n=5) compared with control HemA (normalized to 100%), mock treated HemA (∼100%), and wild-type (2.5%) mice on days 35, 118 and 160, respectively. Additionally, there was neither detectable FVIII activity nor anti-FVIII antibodies in blood on day 160, which indicated that there was insignificant leaky expression of FVIII in other cells. Finally, we also infused G-F8-LV into HemA inhibitor mice. Inhibitors were induced by repeated injection of 3U recombinant hFVIII. The average antibody level was 80 Bethesda Unit before IO infusion of the vectors. In G-F8-LV treated mice, the average hFVIII antigen level on day 27 was 0.74 mU per 1 x 108 platelets (n=5). Bleeding assay was performed on day 160. The blood loss of treated mice was significantly reduced compared with untreated HemA mice, indicating that IO infusion of G-F8-LV can overcome anti-FVIII antibodies and correct hemophilia phenotype. Conclusion We have successfully transduced HSCs in situ by a single infusion of LVs into bone marrow to correct hemophilia A. Gp1bα promoter in lentiviral vectors can specifically direct the transgene expression in mouse platelets. Following IO infusion of G-F8-LV, FVIII stored in platelets can persistently and partially correct the HemA phenotype for at least five months (experimental duration) in mice with and without pre-existing inhibitors. Overall, direct transduction of bone marrow cells targeting platelet-specific FVIII expression may provide an effective therapy to treat severe hemophilia A patients with high-titer inhibitors. Disclosures: No relevant conflicts of interest to declare.

Abstract 3157 Proviral integration into hematopoietic stem cells (HSC) by lentivirus vector (LV)-mediated gene transfer can provide the benefit of life-long therapeutic effect, yet it can also bring the risk of insertional oncogenesis. Platelets are terminally differentiated, enucleated cells full of secretory granules. Restricting transgene expression to platelets may reduce the risk for activating oncogenes in HSCs and most of their progeny, and take advantage of their rapid turnover and professional secretion function. In this study, we evaluated the feasibility of employing megakaryocyte-platelet as a depot for the over-expression and release of alpha-L-iduronidase (IDUA), the lysosomal enzyme deficient in patients with Mucopolysaccharidosis type I (MPS I, or Hurler Syndrome). Utilizing a human megakaryocytic DAMI cell line, we found that a hybrid human ankyrin-1 promoter (K) containing ALAS2 intron 8 enhancer and HS40 core element from human alpha LCR could introduce robust intracellular IDUA expression (over 500-fold of background) and significant enzyme release (120-fold). Upon introduction to megakaryocytic differentiation as verified by FACS analysis with PI staining and morphologic evaluation, the elevated intracellular IDUA levels remained unchanged, while released IDUA increased steadily by 30-fold. We then evaluated in vivo IDUA production/release from platelets in an enzyme-deficient MPS I mouse model. Lineage-negative bone marrow (BM) cells were isolated from MPS I mice using immunomagnetic cell sorting, and transduced twice with LV-K-IDUA-ires-GFP (KIiG) for a total multiplicity of infection at 30. Five-months after 1° transplantation, GFP+ platelets were detected by FACS analysis with CD41-PE staining and observed by Image X analysis. To generate animals with various transgene-dosages, 2° transplantation was performed into MPS I mice using a serial dilution of BM from 1° MPS/KIiG (51–65% transduction efficiency) with BM of MPS I. FACS analysis revealed that the percentage of GFP+ platelets was correlated directly to transduction efficiencies in 2° recipients as determined by real-time qPCR of BM 5 months post transplantation. Moreover, the intra-platelet IDUA enzyme activities were associated linearly with the changes of GFP+% platelets (r2=0.95) among MPS I mice with different transgene-dosages, suggesting that the lysosomal IDUA from transgene overexpression could be sorted and packaged into platelets with proper catalytic function. Noticeably 1% gene transfer efficiency as shown by 1% GFP+ platelets was sufficient to introduce the IDUA levels comparable to those found in wild-type platelets. To determine if platelet-derived IDUA could be released, we conducted Ca-induced platelet activation as verified by FACS analysis with Annexin V-APC and CD41-PE staining, resulting in linear correlation (r2=0.98) of released IDUA with intra-platelet IDUA. Interestingly, the plasma IDUA levels were also linearly correlated with GFP+% platelets (r2=0.96) among MPS I with different transgene-dosages. Competitive uptake assay using lymphoblastoid cells derived from a MPS I patient (LCLmps) demonstrated steady increase of intracellular IDUA with increasing amounts of extracellular enzyme levels, which was blocked by the presence of mannose-6-phosphate (M6P). This suggested that platelet-released IDUA retained its intercellular trafficking capability via M6P receptor. Finally normalization of aberrant lysosomal morphology was observed in LCLmps exposed to platelet-released IDUA as determined by in situ immunostaining with LysoTracker, indicating functional proficiency of platelet-derived IDUA to cross-correct deficit in patient's cells. These results demonstrate for the first time that megakaryocytes/platelets are capable of over-producing, packaging and storing a lysosomal enzyme which retains proper catalytic activity, lysosomal enzyme trafficking and endogenous M6PR-mediated uptake, as well as the ability of cross-correction in patient's cells. This data warrants further evaluation for the potential application of megakaryocytes/platelets in treating lysosomal storage diseases, especially for those, such as Gaucher disease, when the desirable enzyme is sensitive to neutral pH in serum and protection of enzyme in platelets may provide continuous, real-time enzyme release by low physiological levels of platelet activation. Disclosures: No relevant conflicts of interest to declare.

Proviral integration into hematopoietic stem cells (HSC) by lentivirus vector (LV)- mediated gene transfer can provide the benefit of life-long therapeutic effect, yet it can also bring the risk of insertional oncogenesis. Restricting transgene expression to late erythroblasts and red blood cells (RBC) may reduce the risk of activating oncogenes in HSC and its offspring in all lineages. In this study, we sought to evaluate the feasibility of redirecting a fraction of robust protein-synthesis machinery in maturing erythroid cells for production of alpha-L-iduronidase (IDUA), the lysosomal enzyme missing in Mucopolysaccharidosis type I (MPS I, or Hurler Syndrome). We first utilized a murine erythroid leukemia (MEL) cell line to compare IDUA expression and release from LVs using human elongation factor 1α promoter (EF), LTR of SFFV (SF), or a erythroid specific hybrid promoter (IHK) containing human ALAS2 intron 8 erythroid specific enhancer, HS40 core element from human alpha LCR and human ankyrin-1 promoter. We have previously shown highly erythroid-specific expression in vivo using this hybrid promoter, which is sustained in secondary transplantation recipient mice and resists proviral silencing (Moreau-Gaudry et al. Blood, 2001; Mohamedali et al, Mol Ther 2004). MEL cells were introduced to differentiation with hexamethylene bis-acetamide, and progressive erythroid differentiation was confirmed by morphologic evaluation in cytospins. Relatively low levels of expression from IHK (5% of SF, and 8% of EF) were observed in non-induced MEL cells; however, it increased steadily during induction and reached a similarly high expression level as those from the SF promoter. Expression from EF promoter reduced significantly, and the levels from SF promoter remained unchanged with erythroid differentiation. A similar pattern was found in IDUA activities released from stably transduced MEL cells during induction. We then evaluated in vivo the systemic IDUA production in an enzyme-deficient MPS I mouse model using erythroid-specific LV. Lineage-negative bone marrow cells (Lin−) were isolated with 92–97% purity by lineage depletion using immunomagnetic cell sorting. After a short pre-stimulation period, Lin− cells were transduced twice with LV-IHK-IDUA-ires-GFP for a total multiplicity of infection (MOI) at 2–20, resulting in up to 71% transduction efficiency. Four-months after transplantation of Lin- cells transduced at high MOI, sustained and higher-than normal plasma IDUA levels were achieved in all treated MPS I mice (1.5 to 8-fold of normal levels) with GFP transgene expression detected in up to 7% of Ter119+ blood cells. To evaluate transgene expression pattern during erythroid differentiation in bone marrow, we defined erythroblast subpopulations by immunostaining with CD71 and Ter119 for enrichment in proerythroblasts (I), basophilic erythroblasts (II), polychromatophilic erythroblasts (III), and orthrochromatic erythroblasts and reticulocytes (IV). GFP expression was predominantly detected in population III (9–17.3%), followed by the most differentiated population IV. To determine transduction efficiency in HSC, spleen colony-forming-cell assay was performed that showed 15–25% GFP+ spleen colonies in 2o BMT recipients. Moreover, long-term systemic metabolic correction was demonstrated by normalized urinary glycosaminoglycan accumulation in all treated MPS I mice. Complete normalization of tissue pathology was observed in liver and spleen, with relatively moderate improvement in brain. These results demonstrate for the first time that a lysosomal enzyme can be produced and secreted successfully and steadily at superphysiological levels in circulation by erythroid cells using tissue-specific LV, with phenotypic correction of Hurler Syndrome in mice. This data warrants further evaluation for the potential application of erythroid-specific vectors in treating non-RBC related diseases.

The KCl cotransporter mediates volume reduction in normal (AA) reticulocytes, and its abnormal regulation in sickle (SS) reticulocytes contributes to cellular dehydration that facilitates Hb S sickling. mRNA for three KCC genes - KCC1, KCC3, and KCC4 - as well as a splicing isoform, KCC1ex1b, is present in reticulocytes (Exp. Hem.2005; 33:624). Western blotting has demonstrated KCC1 in human RBC membranes (Su et al, AJPhysiol.1999;277:C899) and KCC3 in sheep (Lauf et al, CompBiochemPhysiol2001;130:499); KCC4 protein was found in hRBC membranes by proteomic analysis (Pasini et al, Blood2006;108:791). We confirm here the presence of KCC3 protein in hRBC via western blotting using an antibody to an exon 3 epitope distal to known N-terminal splicing sites. We sought to characterize and compare human KCC isoforms expressed in human cells (HEK 293) and assess their similarity to KCC activity in RBC. cDNAs for human KCC1, KCC1ex1b, KCC3a, and KCC4, with N-terminal c-myc epitope tags were expressed in HEK 293 cells. Stable cell lines were selected by growth in neomycin, and expression monitored by quantitative PCR analysis of the expressed construct and other endogenous isoforms and by western blotting (anti-myc) of plasma membranes. KCC activity was measured as N-ethylmaleimide(NEM)-stimulated, Cl-dependent Rb uptake in cells grown to 75–90 % confluency. Cells were incubated at 37°C in isotonic saline media with various concentrations of Rb (Na replacement), plus 0.1 mM ouabain and 0.01 mM bumetanide. At 2 and 4 min cells were washed with iced Rb-free media, then lysed and assayed for Rb by flame emission, normalized to sample protein. Flux rates were calculated from Rb uptake at 2 and 4 min. The flux rate in Cl-free sulfamate media was subtracted from that in Cl-media to yield the Cl-dependent flux rate. Wild-type HEK cells showed no increase in Cl-dependent Rb influx when exposed to 1 mM NEM, but Cl-dependent, NEM-stimulated Rb uptake was apparent in cells expressing KCC isoforms. All isoforms were stimulated by hypotonic conditions (75 mOsm), but relative to NEM-stimulated activity, KCC3 was most responsive. Kinetic characteristics of Cl-dependent, NEM-stimulated Rb influx in HEK cell expressing human KCC1, KCC3a, and KCC4 isoforms isoforms are given below, compared to RBC KCC fluxes. A splice variant, KCC1ex1b, coding for a protein with a truncated N-terminus, exhibited 48 ± 7% of the activity of the full-length KCC1 isoform, but did not alter transport activity of coexpressed KCC3a. Thus, KCC isoforms expressed in HEK cells share some, but not all, characteristics with RBC KCC fluxes. The isoform kinetics also differ from those previously published in xenopus oocyte expression systems, suggesting that the membrane milieu in which KCC proteins are expressed may influence their functional characteristics. Likewise, KCC activity in RBC may represent an average of several transporters operating in parallel. The truncated KCC1ex1b isoform, expressed at higher levels in normal than in sickle reticulocytes, has lower activity than KCC1. KCC3 and KCC4 exhibit more robust transport activity than KCC1 and may mediate a substantial part of KCC activity in RBC. KCC Kinetic Characteristics VMax (μmol/mgprot./min) Km (ext. Rb, mM) Anion Selectivity hKCC1 28.9 ± 5.6 16.7 ± 5.4 Cl > Br > I > SCN hKCC3a 107 ± 45 11.0 ± 7.1 Cl = Br > I > SCN hKCC4 206 ± 16 15.6 ± 8.2 Cl > Br >> I = SCN RBC NA 12 - 20 Cl = Br >> I = SCN

HIV-based lentiviral vectors (LV) have become powerful tools for in vivo gene transfer and gene expression in non-dividing cells after local injection. However, the potential of in vivo bone marrow stem cell gene transfer by “in situ” bone cavity injection has not been well unexplored. This in vivo approach could take full advantage of any source of stem cells present in bone cavity and avoid many of the difficulties encountered by ex vivo hematopoietic stem cell (HSC) gene transfer. To determine if intra bone marrow (iBM) injection of LV can efficiently transduce BM stem cells, a 3rd-generation self-inactivating LV, containing eGFP regulated transcriptionally by a CMV promoter, was used to inject intra-femorally into adult Boy/J Ly45.1 mice (at 2 x106 IU/mouse). Blood counts were normal in all LV-injected mice (n=4) and buffer-injected mice (n=4). GFP-expressing peripheral blood leukocytes (PBL) were observed in both myeloid (up to 4%) and lymphoid subpopulations (up to 2%) 3-month post injection. To evaluate gene transfer and transgene expression in multi-potential progenitors, the colony-forming cell (CFC) assay was performed using low-density bone marrow 4-months post injection, resulting in 4–5% GFP+ CFU-granulocyte macrophage (CFU-GM) and CFU-granulocyte, erythroid, macrophage and megakaryocyte (CFU-GEMM). This was consistent with the 4-color FACS analysis data demonstrating that up to 3.7% of HSC/progenitors (Lin−c-kit+Sca1+ cells) from LV-injected mice expressed GFP transgene 4-months post injection. To further evaluate HSC gene transfer, BM from injected mice was transplanted into lethally irradiated adult C57/Bl6 (Ly45.2) mice. Four months later, successful engraftment was demonstrated in all BM recipients with 97–99% donor-derived cells in PBL and 91–93.3% in BM. Moreover, significant levels of GFP+ CFU were observed in all recipients ranging from 8.4 ± 2.3% to 17.7 ± 4.2%. To further study the LV-mediated gene transfer profile in HSC, mesenchymal progenitors and systemic distribution by intra-femoral injection, molecular analyses are in progress including real-time QPCR and clonality analysis by LAM-PCR. Our results indicate successful LV-mediated gene transfer into HSC by iBM injection. These data would potentially open a door to a novel approach for treatment of human diseases, but also provide a new tool to study adult stem cell plasticity and the nature of hematopoiesis.