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TRANSLATIONAL AND CLINICAL RESEARCH |
aDepartment of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada;
bDepartment of Pediatrics, Nara Medical University, Kashihara City, Nara, Japan;
cVascular Biology Center and Division of Hematology Oncology Transplantation, University of Minnesota Medical School, Minneapolis, Minnesota, USA;
dLady Davis Institute for Medical Research, McGill University, Montreal, Quebec, Canada
Key Words. Hemophilia A • Lentivirus • Blood outgrowth endothelial cells • Ex vivo gene therapy
Correspondence: David Lillicrap, M.D., Department of Pathology and Molecular Medicine, Richardson Laboratory, Queen's University, Kingston, Ontario, Canada K7L 3N6. Telephone: 613-548-1304; Fax: 613-548-1356; e-mail: lillicrap{at}cliff.path.queensu.ca
Received on November 1, 2006;
accepted for publication on June 20, 2007.
First published online in STEM CELLS EXPRESS July 5, 2007.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Hemophilia continues to be an excellent candidate disease for the application of gene transfer [2–5], and significant advances and success in preclinical testing in animal models of hemophilia A have resulted in several small, phase 1/2 human clinical trials. However, because of only transient low-level FVIII expression, none of these trials has progressed further [6–7].
Most preclinical studies for hemophilia gene therapy have focused on systemic delivery of viral vectors for in vivo transduction of liver cells [8]. Several such studies have yielded promising results, and long-term therapeutic levels of plasma FVIII in hemophilia A mice have been achieved by systemic delivery of adenoviral, adeno-associated viral (AAV), oncoretroviral, and lentiviral vectors [9–17]. However, there remain concerns over the safety of these approaches. Potential side effects include adverse immunological reactions, vector-mediated cytotoxicity, germ-line transmission, and oncogenesis [18–22]. Ex vivo strategies for gene therapy could possibly circumvent some of these obstacles. Since FVIII is a secreted protein and its expression is not limited to hepatocytes, other somatic cells might be candidates to target for ex vivo gene therapy strategies [23–26]. Indeed, the implantation of ex vivo modified autologous fibroblasts that secreted FVIII is well tolerated and leads to transient increments of plasma FVIII in patients with hemophilia A [27].
Although there has been interest in the use of blood outgrowth endothelial cells (BOECs) for vascular regeneration [28], these cells may also be an alternative target cell for ex vivo strategies of FVIII gene therapy. In a previous preclinical study, human BOECs were modified ex vivo to express human FVIII and introduced systemically into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. This resulted in the expression of therapeutic levels of plasma human FVIII in the treated animals [29].
In this study, we have further explored the therapeutic potential of genetically modified BOECs by evaluating their use, for the first time, as a subcutaneous depot for FVIII delivery. We report sustained therapeutic levels of FVIII for up to 20 weeks in both NOD/SCID and immunocompetent hemophilia A mice.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Production and Concentration of Lentiviral Vectors
The lentiviral vectors Lenti-CMV-cFVIII, Lenti-CMV-GFP, Lenti-TM-GFP, and Lenti-TM-cFVIII were prepared by transient transfection of 293T cells with either pLenti-CMV-cFVIII, pLenti-CMV-GFP, pLenti-TM-GFP, or pLenti-TM-cFVIII, as well as pMDLgag/pol RRE, pRSV-Rev, and pCI-VSV-G. The following amounts of DNA were used per 15-cm plate: 22.86 µg of lenti-vector plasmid, 30.71 µg of pMDL gag/pol, 61.43 µg of pRSV-Rev, and 38.86 µg of pCI-VSV-G. 293T cells were cultured in D10 medium, which consists of Dulbecco's modified Eagle's medium supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml, streptomycin, and 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich, Oakville, ON, Canada, http://www.sigmaaldrich.com). Cells were grown in culture plates coated with poly-L-lysine. When cells were 80%–90% confluent, the plasmids were transfected by the Chen-Okayama calcium phosphate transfection protocol [32]. One hour before transfection was initiated, the medium was changed to fresh D10 medium supplemented with sodium butyrate (final concentration, 4 mM; Sigma-Aldrich). At 24 hours post-transfection, the medium with floating dead cells was removed, cells were washed with phosphate-buffered saline (PBS), and 25 ml of OptiMEM I (Invitrogen) was substituted as culture medium [33]. Conditioned medium was collected every 24 hours during the subsequent 2 days. The filtered vector supernatant was concentrated by ultracentrifugation at 20,000 rpm for 140 minutes using a Beckman SW28 rotor (Beckman Coulter, Mississauga, ON, Canada, http://www.beckmancoulter.com). The supernatant was discarded, the pellet was resuspended in 1.5 ml of sterile PBS, and a repeat centrifugation was carried out. The pellet, which originated from 300 ml of conditioned medium, was resuspended in 800 µl of PBS. Concentrated virus was stored at –80°C.
Vector Titering
The functional titer of lentiviral vectors was determined as described previously [34] with minor modification. Briefly, 293T cells were seeded in a six-well plate (1 x 105 cells per well) and transduced with serially diluted vector supplemented with 8 µg/ml polybrene (Sigma-Aldrich). After 8 hours of incubation, the medium was replaced with fresh D10 medium. Cells were harvested 72 hours after transduction, and DNA was extracted as previously described [35]. The copy number of integrated lentiviral vector sequences, relative to the β-actin gene from the 293T cell genome, was assessed by quantitative real-time PCR. Vector-derived sequence was amplified using 5'-ACCTGAAAGCGAAAGGGAAAC-3' sense and 5'-CACCCATCTCTCTCCTTCTAGCC-3' antisense primers and a probe LV2, 5'-FAM-AGCTCTCTCGACGCAGGACTCGGC-MGB3' (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) [29]. Ampligold TaqMan Master Mix (Applied Biosystems) was used for amplification reactions carried out with an ABI Prism 7000 sequence detection system. Reaction conditions were as follows: one cycle of 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Various amounts of lentivector plasmid DNA were used to generate a standard curve. Reactions were carried out separately to measure β-actin as the endogenous reference. The probe β-actin, 5'VIC-TGGCACCACACCTT-MGB3' (Applied Biosystems), and primers 5'-CAACTGGGACGACATGGAGAA-3' (sense) and 5'-GCCACACGCAGCTCATTGTA-3' (antisense) were homologous to the mouse β-actin gene. The PCR fragment containing the β-actin sequence was subcloned into pCR2.1 (Invitrogen), and various amounts of this construct were used to derive a standard curve. The integrating copy number represents the vector titer expressed as infectious units per milliliter and was determined as follows:
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Canine and Murine BOEC Isolation
Canine BOECs were isolated from venous blood obtained from a normal dog, as previously described [28, 29]. In brief, we obtained buffy coat mononuclear cells from 90 ml of freshly isolated blood using Histopaque 1077. After suspending these cells in MCDB131 medium (Gibco, Grand Island, NY, http://www.invitrogen.com) supplemented with endothelial cell growth medium-2 (Clonetics, San Diego, http://www.cambrex.com), 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10% FBS, we plated them in a bovine type I collagen-coated 12-well plate. Approximately 2–3 weeks later, small colonies of cells were visualized that were confluent within 10 days. The cells had typical endothelial cobblestone morphology and expressed von Willebrand factor (VWF) but not FVIII. The cells had undergone 3–4 cell passages at the time of use for these experiments. Murine BOECs were isolated from the pooled blood of 15 C57BL/6 hemophilia A mice (approximately 20 ml) using an approach similar to that described for canine cells.
Lentiviral Vector Transduction of BOECs In Vitro
Cultured canine BOECs (1 x 105) were transduced following a single exposure to the Lenti-CMV-GFP, Lenti-CMV-cFVIII, Lenti-TM-GFP or Lenti-TM-cFVIII viral vectors at increasing multiplicities of infection (MOIs). After transduction, cells were expanded as previously described. Transduction efficiency was evaluated with cells transduced with Lenti-CMV-GFP or Lenti-TM-GFP by flow cytometry using an EPICS ALTRA HSS analyzer (Beckman Coulter). Assessment of FVIII expression from BOECs transduced with Lenti-CMV-cFVIII or Lenti-TM-cFVIII was carried out using a functional chromogenic assay (DiaPharma, West Chester, OH, http://www.diapharma.com), as directed by the manufacturer, to determine FVIII activity in the cell culture medium. Pooled normal human plasma was used to establish the FVIII standard curve (Precision Biologicals, Dartmouth, NS, Canada). The sensitivity of this functional FVIII assay is 10 mU/ml.
Animal Procedures
Nonhemophilic NOD/SCID mice were obtained from Taconic Farms, Inc. (Hudson, NY, http://taconic.com), and immunocompetent hemophilia A mice were a kind gift from Dr. H.H. Kazazian (University of Pennsylvania). All animal procedures were reviewed and approved by the Queen's University Animal Care Committee. BOECs transduced with Lenti-CMV-cFVIII, Lenti-CMV-GFP, Lenti-TM-GFP, or Lenti-TM-cFVIII were trypsinized and concentrated by centrifugation. Three consecutive i.v. tail vein injections each of 2 x 106 cells in 300 µl of PBS were infused daily over a 3-day period to 3-week-old NOD/SCID mice. For the subcutaneous implantation studies, 2 x 106 BOECs, resuspended in 300 µl of PBS, were mixed with 500 µl of Matrigel (BD Biosciences, Bedford, MA, http://www.bdbiosciences.com) and implanted by subcutaneous injection into the center back of the neck of 3-week-old NOD/SCID or hemophilia A mice. Matrigel is a solubilized murine basement membrane preparation in which the major constituents are laminin and type IV collagen. Identical procedures were used for control mice but, instead, nontransduced BOECs were administered.
Immunocompetent hemophilia A mice were also treated with subcutaneously implanted, genetically modified BOECs. Six of these mice received the subcutaneous implants without any prior treatments; four mice received the implants following the prior administration of two i.v. injections of recombinant canine FVIII (20 unit/kg per injection) [36], 5 and 2 days prior to the implant, and three mice were treated following the intraperitoneal injection of cyclophosphamide (20 mg/kg per injection) administered on the day of the implantation and then biweekly for 4 weeks.
Biodistribution of BOECs in Mice That Received Lenti-CMV-GFP-Transduced Cells by Tail Vein Infusion or by Subcutaneous Implantation
Four and 12 weeks postadministration, NOD/SCID mice were sacrificed, and multiple organs as well as the subcutaneous implant were harvested. Mouse tissues were homogenized with the end of a sterile syringe placed in a 70-µm cell strainer (BD Biosciences) over a 50-ml conical tube. The cells were centrifuged at 1,500 rpm for 5 minutes, and the red blood cells were lysed with Tris-buffered ammonium chloride. The cells were separated again, and the pellet was resuspended in PBS/1.5% paraformaldehyde. Flow cytometric analysis was used to count and analyze the percentage of GFP-positive BOECs in each tissue sample. Retrieved Matrigel implants were cut into approximately 2-mm3 fragments and treated with 1.6 mg/ml type IV collagenase (Sigma-Aldrich) and 200 µg/ml DNase (Sigma-Aldrich) in PBS at 37°C for 1 hour. A total of 1 x 106 cells were analyzed for GFP expression as outlined for the mouse tissue samples.
Quantification of FVIII Levels in Mouse Plasma
Blood samples were first collected 2 weeks after injection from the saphenous vein and subsequently at weekly intervals. FVIII antigen levels were assayed using an Asserachrom VIII:Ag (antigen) enzyme-linked immunosorbent assay (ELISA) (Diagnostica Stago, Asnieres-Sur-Senie, France, http://www.stago-us.com) that detects canine FVIII against a background of normal murine FVIII levels in the NOD/SCID mice. The standard curve for this assay was generated with pooled normal canine plasma, and the sensitivity of the assay is 10 mU/ml (1% FVIII level). In the hemophilia A mice, functional FVIII was quantified by a chromogenic bioassay as described above. The development of anti-FVIII antibodies in hemophilia A mice was detected and quantified by the Bethesda assay [37].
Subcutaneous Implant Removal and Immunohistochemical Analysis
After 3, 8–10, 18, or 20 weeks postimplantation, NOD/SCID mice that had embedded subcutaneous implants were sacrificed. The implants were recovered, fixed with 10% formalin, and embedded in paraffin. To assess FVIII expression in BOECs transduced with Lenti-CMV-FVIII within the retrieved Matrigel implants, BOECs were characterized with triple immunostaining for VWF, FVIII, and cell nuclei. A rabbit antibody to human VWF (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com) was used to detect the canine VWF, and a porcine fluorescein isothiocyanate-labeled anti-rabbit immunoglobulin (DakoCytomation) served as the detecting antibody. For FVIII staining, we used a sheep anti-canine FVIII antibody (Affinity Biologicals, Ancaster, ON, Canada, http://www.affinitybiologicals.com) that was labeled with Cy3 using the Flulolink Ab Cy3 labeling kit (Amersham Biosciences, Buckinghamshire, U.K., http://www.amersham.com) according to the manufacturer's instructions. Cell nuclei were stained with 4,6-diamidino-2-phenylindole. Specimens were viewed with a confocal laser scanning microscope (Leica TCS SP2 multiphoton; Leica, Richmond Hill, ON, Canada, http://www.leica.com).
Quantification of Vector Copy Number in Mouse Organs
The integrated vector copy number in transduced mouse tissues was also determined by quantitative PCR as detailed above. Animals were euthanized by CO2 inhalation. Organs were collected and DNA extracted as previously described. The integrating copy number of lentiviral vectors was determined with the use of the LV2 probe. The endogenous β-actin gene was used to normalize for the amount of DNA.
Statistical Analysis
The data are presented as mean ± SEM. Statistical comparisons of the biodistribution of BOECs between i.v. delivery and subcutaneous injection into Matrigel was carried out using a Fisher's exact test. A p value of less than 0.05 was considered statistically significant.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Analysis of the Biodistribution of Engineered BOECs Administered to NOD/SCID Mice
To investigate the potential use of genetically modified BOECs in an ex vivo strategy for hemophilia A gene therapy, we initially assessed the biodistribution and viability of Lenti-CMV-GFP transduced canine BOECs at various MOIs administered via the tail vein or implanted subcutaneously in Matrigel. To avoid an inevitable xenogeneic immune response, the engineered canine BOECs were administered to NOD/SCID mice. Similar to recently published data [38], in all four mice that received engineered BOECs via a tail vein injection, GFP-positive cells were predominantly isolated from the liver, lung, and bone marrow, with lower levels of GFP-positive cells observed in the heart and spleen (Table 1). By 12 weeks postinjection, there was a substantial reduction in the relative percentage of GFP-positive cells analyzed from comparable tissues.
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Long-Term In Vivo cFVIII Antigen Expression from Genetically Engineered BOECs Administered to NOD/SCID and Immunocompetent Hemophilia A Mice
To compare the efficacy of the two ex vivo strategies to produce therapeutically relevant levels of FVIII, Lenti-CMV-FVIII transduced BOEC at various MOI were initially administered to NOD/SCID mice via the tail vein or implanted subcutaneously in Matrigel (Table 2), and plasma levels of cFVIII antigen were assayed using the Asserachrom FVIII:Ag ELISA. As observed in Figure 2A, the mean cFVIII antigen rose from 0 to 37.5 ± 12.4 mU/ml (mean ± SEM) by 3 weeks after i.v. injection of Lenti-CMV-cFVIII-transduced BOECs, and therapeutic levels (
20 mU/ml) persisted for 12 weeks (21.3 ± 0.4 mU/ml). Control mice injected with nontransduced BOECs showed baseline cFVIII levels over the duration of the experiment. In contrast, the mean levels of plasma cFVIII antigen rose from 0 to 105.8 ± 62.3 mU/ml 3 weeks after subcutaneous injection of BOECs mixed with Matrigel (Fig. 2B) and remained at therapeutic levels until 12 weeks (21.7 ± 0.2 mU/ml) before declining to pretreatment baseline.
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To determine whether plasma levels of FVIII and VWF could be further increased, 1-deamino-8-D-arginine-vasopressin (DDAVP) (50 µg/kg/mouse) was administered via the tail vein to two normal, untreated mice and two mice that had received a subcutaneous injection of Lenti-TM-cFVIII engineered BOECs in Matrigel. No differences were observed in the levels of plasma FVIII or VWF 2, 24, and 168 hours after DDAVP infusion in any of these mice.
Although prolonged expression of FVIII in NOD/SCID mice demonstrates the proof-of-principle for this treatment strategy, the immunocompromised status of these animals does not allow for any evaluation of an immune response to the delivery system or the transgene product. We therefore repeated these studies with the optimal delivery protocol using the thrombomodulin-regulated construct with subcutaneous implantation of syngeneic murine BOECs in immunocompetent hemophilia A mice.
Six hemophilia A mice were treated with the BOEC implants without any prior treatments. None of these six mice showed evidence of FVIII expression, but by 8 weeks, all of the mice had developed neutralizing anti-FVIII antibodies between 5 and 25 Bethesda units in titer (Fig. 3A). In contrast, four mice treated with two tolerizing i.v. doses of canine FVIII protein (20 U/kg) and three mice treated with peri-implantation cyclophosphamide (20 mg/kg given i.p.) all expressed FVIII at therapeutic levels between 20 and 30 mU/ml for up to 27 weeks (Fig. 3B, 3C). None of these mice developed anti-FVIII inhibitory antibodies. After 27 weeks, plasma FVIII levels began to drop and reached undetectable levels in all mice by 30 weeks.
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Quantification of Vector Copy Number in Mouse Organs After BOEC Administration
Since lentiviral vectors integrate into the genome of transduced BOECs, it is possible to identify the engineered BOECs either in the subcutaneous implant or distributed throughout the various organs by using quantitative real-time PCR (Q-PCR) analysis for the presence of lentiviral sequences. An estimate of the lentiviral copy number was determined in various NOD/SCID mice 3, 8–10, or 20 weeks after transduced BOECs were administered intravenously (Fig. 4A) or subcutaneously in the Matrigel scaffold (Fig. 4B). Vector copy numbers are represented relative to the β-actin sequence. In the BOECs delivered by i.v. injection, copies of the lentiviral DNA were detected in all tested organs, with the highest copy number in liver (transgene copy number relative to β actin of 0.32). However, the relative copies of the vector DNA decreased by approximately 10-fold over the 15 weeks of this experiment. These data confirm that i.v. delivery of engineered BOECs results in widespread biodistribution with gradual loss of the cells over time. However, in the mice treated with subcutaneously implanted BOECs, copies of lentivirus DNA were detected only in the Matrigel implant and not in any of the other analyzed organs or tissues (0.42 copies of the transgene relative to the endogenous β actin). In the subcutaneous implants, the reduction in vector was more (60-fold) than the reduction in β-actin (23-fold) over 15 weeks, and the overall relative reduction in lentiviral copy number was only 2.5-fold, compared with a 10-fold loss in tissues after i.v. administration. These data support the results of the aforementioned GFP flow cytometry analysis and provide additional evidence that the genetically modified BOECs are retained exclusively inside the subcutaneous implant. The relative copy number of lentiviral DNA inside the subcutaneous implant decreased over time, presumably in part from some form of cell death of the engineered BOECs. However, the relative number of implanted canine BOECs could also be reduced in part by an influx of host cells.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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In this study, we sought to investigate an alternative gene transfer strategy that would be safer and that would result in sustained FVIII transgene expression. We used circulating endothelial precursor cells (BOECs) that were transduced ex vivo with lentiviral vectors containing cFVIII transgenes regulated by either the viral CMV or the endothelial TM promoters. The engineered cells were implanted subcutaneously in a soluble basement membrane scaffold, thereby enhancing the safety of the strategy since the gene-modified cells are sequestered from the rest of the body. Long-term FVIII expression of 20–30 mU/ml has been achieved in both immunocompromised and immunocompetent mice, a level of FVIII expression that has been shown from protein replacement studies to provide effective prophylaxis against bleeding [44].
A major challenge in the development of ex vivo gene transfer strategies for hemophilia A has been the achievement of sustained production of therapeutic levels of FVIII. This requires the stable delivery of a FVIII transgene to cells that have a long life span and/or cells with long-term repopulation capacity. In this study, we chose to use lentivirally transduced BOECs as the cell for FVIII transgene expression. BOECs are easy to isolate and propagate from blood and have the potential for both extended longevity and the generation of daughter endothelial cells [28], both ideal characteristics for sustained gene transfer. Furthermore, lentivectors efficiently transduce these cells and stably integrate into the progenitor cell genome. In this study, the Lenti-CMV-cFVIII transduced BOECs were able to produce high concentrations of functionally active FVIII in vitro. These levels were in the order of 10-fold higher than those in a similar ex vivo study where human BOECs were transfected, via lipofection, with a CMV-regulated human FVIII expression plasmid [29]. In this earlier study, the i.v. administration of selected, stably transfected human BOECs into NOD/SCID mice at cell doses between 1.5 x 105 and 1.2 x 106 cells per mouse (compared with 2–6 x 106 cells per mouse in the current study) resulted in sustained levels of plasma FVIII that were substantially higher (approximately 50-fold) than those documented in the current report. However, in this previous study, these higher levels of FVIII were achieved only under the following circumstances: the best-expressing BOEC clones had to be isolated with a selectable marker; the transfected BOECs were delivered systemically by i.v. injection, resulting in widespread dissemination of the cells; and there was evidence of at least a 10-fold expansion of the BOECs following in vivo delivery. We would propose that each of these factors complicates the use of genetically modified BOECs for gene transfer and also raises concerns about systemically administered progenitors as a source for transgene expression.
Another potential benefit that might accrue from using BOECs to express FVIII is the well-characterized protective effect that VWF exerts over FVIII in plasma. Although a very recent publication has documented synthesis of FVIII from pulmonary endothelial cells [45], the native coincident expression of these two proteins in endothelium has not been evaluated. In this report, our limited immunostaining studies suggest that approximately 30% of the two proteins colocalize within the transduced BOECs. These results appear to be similar to those recently obtained with the transgenic expression of FVIII in platelets, in which colocalization is seen for some, but not all, of the two proteins [46].
Previously, we and other groups have observed that systemic delivery of engineered BOECs resulted in the transplantation of these cells into several different organs [29]. As discussed above, a significant concern with the transduction of stem cells with a lentiviral or retroviral vector is the possibility of insertional mutagenesis and subsequent oncogenesis [22, 41, 42]. Analysis of the biodistribution of the genetically modified BOECs expressing GFP and cFVIII revealed no evidence of these gene-modified cells at other sites. These results indicate that the basement membrane scaffold used for the subcutaneous implants is very effective at sequestering the cells at this anatomic site, thus avoiding the disseminated delivery and significant expansion of cells seen with i.v. injection of genetically modified progenitor cells [29]. Although we have not carried out a detailed assessment of cell numbers, there was no evidence, in the current study, of any significant in vivo expansion of the subcutaneous BOEC implants. This gene transfer approach might also lend itself to the removal and replacement of the implant should this be deemed necessary. Although in other species, the mouse Matrigel scaffold is likely to incite an immunologic response, alternative synthetic matrices are now available that will likely not be immunogenic but will still effectively sequester the implanted cells.
In this study, therapeutic levels of plasma FVIII were documented from the Lenti-CMV-cFVIII engineered BOECs delivered to NOD/SCID mice for 12 weeks. Since these mice lack functional T and B cells, the loss of FVIII expression cannot be attributed to FVIII-specific antibodies. Immunostaining of the Lenti-CMV-cFVIII engineered cells retrieved from the subcutaneous implant shows a gradual loss of VWF and FVIII expression over the first 2 months following implantation. Moreover, quantitative PCR to determine the viral copy number also indicated a gradual loss of vector DNA in the implanted cells. Overall, these results suggest that the loss of FVIII expression is due, at least in part, to a gradual loss of BOEC viability. In this study, we have demonstrated that the use of the native thrombomodulin promoter extends the duration of in vivo expression of FVIII from genetically modified cells from 12 weeks to at least 27 weeks. At 27 weeks, there was a marked decline in plasma FVIII, and by 30 weeks no FVIII was detected in any of the mice. Since no Matrigel implant or engineered BOECs could be detected at 35 weeks, it is unclear whether the loss of plasma FVIII resulted from a breakdown of the scaffold material of from BOEC death. Studies are ongoing to evaluate alternative scaffold materials and strategies to enhance the viability of the implanted BOECs.
In addition to demonstrating persistent therapeutic expression of FVIII in the NOD/SCID model, we have also demonstrated similar long-term FVIII expression in immunocompetent hemophilia A mice. However, as we had expected, these results were obtained only following the prior administration of tolerizing doses of canine FVIII or with transient peri-implantation immunosuppression. Without these maneuvers, every mouse developed an anti-FVIII humoral immune response.
The results presented here show, for the first time, that genetically modified BOECs are viable in a subcutaneous implant, that they remain sequestered at this site, and that they are capable of synthesizing therapeutic levels of plasma FVIII over a prolonged time period. This novel ex vivo gene transfer strategy can provide for the safe and efficacious delivery of FVIII in hemophilia A and merits further assessment.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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REFERENCES |
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