Sustained phenotypic correction of hemophilia a mice following oncoretroviral mediated expression of a bioengineered human factor viii gene in long term hematopoietic repopulating cells

Sustained phenotypic correction of hemophilia a mice following oncoretroviral mediated expression of a bioengineered human factor VIII gene in long term hematopoietic repopulating cells ARTICLE doi 10[.]

ARTICLE doi:10.1016/j.ymthe.2004.08.006 Sustained Phenotypic Correction of Hemophilia A Mice Following Oncoretroviral-Mediated Expression of a Bioengineered Human Factor VIII Gene in Long-Term Hematopoietic Repopulating Cells Morvarid Moayeri,1,2 Ali Ramezani,1 Richard A Morgan,3 Teresa S Hawley,4and Robert G Hawley,1,2,* Department of Anatomy and Cell Biology and 4Flow Cytometry Core Facility, The George Washington University Medical Center, Washington, DC 20037, USA 2Graduate Genetics Program, The George Washington University, Washington, DC 20052, USA Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA *To whom correspondence and reprint requests should be addressed at the Department of Anatomy and Cell Biology, The George Washington University Medical Center, Suite 419, 2300 Eye Street, NW, Washington, DC 20037, USA Fax: (202) 994–8885 E-mail: rghawley@gwu.edu Available online September 2004 Hematopoietic stem cells (HSCs) are an attractive target cell population for hemophilia A gene therapy because of their capacity to regenerate the hematolymphoid system permanently following transplantation Here we transplanted bone marrow (BM) cells transduced with a splicing-optimized MSCV oncoretroviral vector expressing a secretion-improved human factor VIII gene into immunocompromised hemophilic mice that had received a reduced dose conditioning regimen An enhanced green fluorescent protein (EGFP) reporter gene linked to an encephalomyocarditis virus internal ribosome entry site was incorporated into the vector to allow preselection of transduced cells and facile evaluation of engraftment Sustained expression of EGFP was demonstrated in the peripheral blood, and therapeutic levels of factor VIII were detected in the plasma of the majority of the recipients for the duration of the observation period (up to 22 weeks) Coordinate expression of factor VIII and EGFP (up to 19 weeks) was transferred to secondary BM transplant recipients, indicating that long-term repopulating HSCs had been successfully gene modified Notably, the hemophilic phenotype of all treated mice was corrected, thus demonstrating the potential of HSC-directed oncoretroviral-mediated factor VIII gene transfer as a curative therapeutic strategy for hemophilia A Key Words: hemophilia A, factor VIII gene therapy, oncoretroviral vector, hematopoietic stem cells INTRODUCTION Hemophilia A is an X-linked recessive bleeding disorder affecting in 5000–10,000 males that is caused by a deficiency or functional defect in coagulation factor VIII [1–4] Patients suffer from frequent spontaneous and trauma-induced joint and soft-tissue hemorrhage, leading to chronic debilitating arthropathy and, in severe cases, death Based on the residual activity of factor VIII in plasma, hemophilia A is categorized as severe (b1% of normal activity), moderate (1–5%), and mild (5–30%) The current treatment is replacement of deficient factor VIII with frequent infusions of plasmaderived or recombinant factor VIII protein Very high cost and unpredictable shortages of recombinant factor VIII and risk of transmission of certain blood-borne 892 viruses (such as hepatitis A, B, and C; HIV; and parvovirus) with plasma-derived factor VIII are among the disadvantages of replacement therapy Furthermore, a very serious complication of this treatment is the development of neutralizing binhibitorQ antibodies against factor VIII in approximately 25% of hemophilia A patients, rendering them unresponsive to further factor VIII protein infusions [2,3] Hemophilia A is an excellent candidate for gene therapy because it is a monogenic disorder, modest elevation of factor VIII levels to 1.5–2% of normal is sufficient to improve significantly the clinical symptoms, and tissue-specific expression is not required [1,4] Hematopoietic stem cells (HSCs) are an attractive target cell population for hemophilia A gene therapy MOLECULAR THERAPY Vol 10, No 5, November 2004 Copyright C The American Society of Gene Therapy 1525-0016/$30.00 doi:10.1016/j.ymthe.2004.08.006 because they are readily accessible and allow for the possibility of long-term expression of an integrated factor VIII transgene from circulating cells in peripheral blood [1] Moloney murine leukemia virus-derived oncoretroviral vectors are widely used for HSC gene transfer because their stable chromosomal integration provides the opportunity for lifelong expression of their encoding transgenes [5] Importantly, recent advances in methodology have resulted in therapeutic efficiencies of oncoretroviral gene transfer to HSCs in preclinical studies of nonhuman primates and in human clinical trials [6–8] Nonetheless, prior attempts to achieve prolonged clinically relevant plasma levels of factor VIII by HSC-directed oncoretroviral-mediated gene delivery approaches were unsuccessful [9,10] In this paper we describe the construction of MSGVsfVIIIDB-IRES-EGFP, a murine stem cell virus (MSCV)based oncoretroviral vector [11] carrying a B-domaindeleted factor VIII cDNA (sfVIIIDB) bioengineered for enhanced secretion The MSCV vector has been demonstrated to direct reliable transgene expression in the reconstituted hematopoietic systems of mice following engraftment with gene-modified HSCs [12] and in the lymphomyeloid progeny of transduced candidate human HSCs assayed in murine xenotransplant models [13,14] Because it had been observed that inclusion of an intron 5Vof the factor VIII cDNA in an oncoretroviral vector led to higher steady-state levels of factor VIII mRNA [15], the MSCV backbone was modified for more efficient splicing of transgene transcripts by incorporation of the extended gag region and env splice site from the MFG vector [16,17] Previous work identified specific mutations in the A1 domain of factor VIII that increased secretion severalfold over that of the wild-type protein [18] Therefore, these mutations were introduced into the sfVIIIDB gene by site-directed mutagenesis To avoid the complications of a potential immune response against factor VIII neoantigen [10,19], an immunocompromised hemophilia A double-knockout mouse strain (E-16 / / B7-2 / ) was utilized [20] Here we report that within this experimental setting, engraftment of minimally myeloablated primary and secondary recipients with MSGVsfVIIIDB-IRES-EGFP-transduced bone marrow (BM) cells resulted in sustained therapeutic plasma levels of sfVIIIDB-encoded factor VIII and long-term correction of the hemophilic phenotype These findings provide an important framework for the development of future hemophilia A gene therapy strategies targeting HSCs RESULTS In Vitro Analysis of Functional Factor VIII Production by MSGV-sfVIIIDB-IRES-EGFP-Transduced Heterologous Cells We transduced murine NIH3T3 fibroblasts with helperfree ecotropic MSGV-sfVIIIDB-IRES-EGFP vector particles MOLECULAR THERAPY Vol 10, No 5, November 2004 Copyright C The American Society of Gene Therapy ARTICLE and sorted them based on expression of an incorporated enhanced green fluorescent protein (EGFP) reporter gene We used these cells to evaluate the integrity of the vector DNA and the abundance of vector transcripts by Southern and Northern blot analysis, respectively We digested genomic DNA with SacI, which cleaves the vector within both long terminal repeats (LTRs) and at the 5V end of the sfVIIIDB transgene (Fig 1A) We used an EGFP probe to detect the sfVIIIDB-IRES-EGFP cassette in the Southern blot analysis The presence of a single 6.4-kb band in the transduced NIH3T3 cells indicated that the majority of the integrated sfVIIIDB transgenes were faithfully transmitted without rearrangement (Fig 1B) We subjected total RNA isolated from transduced and control NIH3T3 cells to Northern blot analysis using the same EGFP-specific probe The Northern blot revealed two major transcripts in transduced but not control cells (Fig 1C), an 8.1-kb band corresponding to full-length vector RNA plus a 7.2-kb band corresponding to spliced sfVIIIDB-IRES-EGFP mRNA as predicted (see Fig 1A) After obtaining evidence of the structural integrity of the sfVIIIDB cDNA and documenting high levels of spliced sfVIIIDB-IRES-EGFP mRNA, we evaluated secretion of sfVIIIDB-encoded protein from transduced NIH3T3 cells by Western blot analysis (Fig 2A) We harvested culture supernatants and extracts from transduced EGFP-sorted and control NIH3T3 cells and performed immunoprecipitations with two factor VIII lightchain-specific monoclonal antibodies (ESH2 and ESH8) Following polyacrylamide gel electrophoresis and transfer to a polyvinylidene difluoride (PVDF) membrane, we identified factor VIII cross-reactive material with an antifactor VIII polyclonal antibody (SAF8C-AP) We used recombinant full-length human factor VIII, comprising primarily a heavy chain migrating at 200 kDa and a light chain migrating at 80 kDa, as a positive control Both chains of recombinant human factor VIII were detected with the SAF8C-AP anti-factor VIII polyclonal antibody although the 80-kDa light chain band was poorly visualized, likely due to the low reactivity of this antibody for light-chain epitopes as suggested previously [19] A strong band of approximately 170 kDa, which corresponded to the primary sfVIIIDB translation product representative of B-domain-deleted factor VIII single chain [19,21], was detected in the lane containing cell extract of MSGV-sfVIIIDB-IRES-EGFP-transduced cells In the conditioned medium from MSGV-sfVIIIDB-IRESEGFP-transduced cells, a faint 170-kDa sfVIIIDB single chain band could be seen along with a prominent doublet of 92 kDa that corresponded to glycosylation variants of processed sfVIIIDB heavy chain species characteristic of B-domain-deleted factor VIII [21] We also observed a prominent 90-kDa band, which probably represents a cleavage product of the sfVIIIDB heavy chain due to the presence of residual thrombin-like activity in 893 ARTICLE doi:10.1016/j.ymthe.2004.08.006 FIG Structure of the MSGV-sfVIIIDB-IRES-EGFP vector and expression in transduced NIH3T3 cells (A) Schematic representation of the MSGV-sfVIIIDB-IRES-EGFP vector indicating the predicted 8.1-kb (full-length vector RNA) and 7.2-kb (spliced sfVIIIDB/EGFP mRNA) transcripts Shown are the SacI restriction sites used for analysis of vector sequence transmission Abbreviations: EGFP, enhanced green fluorescent protein gene; IRES, internal ribosome entry site; LTR, long terminal repeat; SA, splice acceptor; SD, splice donor; sfVIIIDB, secretion-enhanced B-domain-deleted factor VIII gene (B) Southern blot analysis Genomic DNA obtained from nontransduced and transduced NIH3T3 cells was digested with SacI and vector structural integrity was assessed by hybridization with an EGFP-specific probe The blot was rehybridized with a probe specific for the endogenous murine bcl2 gene to monitor completeness of DNA digestion (C) Northern blot analysis Total RNA was extracted from nontransduced and transduced NIH3T3 cells and vector transcripts were detected by hybridization with an EGFP-specific probe The blot was rehybridized with a probe specific for hactin sequences to monitor intactness of the RNA the conditioned medium [21,22] As found for the recombinant factor VIII light chain, the mature sfVIIIDB light chain, which migrated as an 80-kDa doublet [21], was only weakly detected by the SAF8C-AP polyclonal antibody albeit still clearly discernible on the blot To determine whether the associated heavy and light chain complex of the secreted factor VIII was biologically active, we analyzed 24-h culture supernatants from transduced NIH3T3 cells using a chromogenic assay (COATEST VIII:C/4) In this assay, factor VIII acts as a FIG Secretion of functional factor VIII from MSGVsfVIIIDB-IRES-EGFP-transduced NIH3T3 cells (A) Western blot analysis Cell extracts and conditioned medium from nontransduced and transduced NIH3T3 cells were immunoprecipitated with two light-chain-specific monoclonal antibodies (ESH2 and ESH8), electrophoresed through a 4–12% Bis– Tris NuPAGE gel under reducing conditions, and blotted onto a PVDF membrane Factor VIII proteins were identified with an anti-human factor VIII polyclonal primary antibody (SAF8C-AP), an alkaline phosphatase-conjugated secondary antibody, and ECF chemifluorescence detection reagent SC, single chain; HC, heavy chain; LC, light chain The heavy and light chains of recombinant human factor VIII used as a positive control are indicated by asterisks (B) Assay for functional sfVIIIDB-encoded factor VIII activity: Twenty-four-hour conditioned medium was collected from nontransduced and transduced NIH3T3 cells Factor VIII activity was measured using the chromogenic functional COATEST assay The bars represent factor VIII concentration corresponding to factor VIII activity calculated using a standard curve and given in mIU/106 cells/24 h All samples were assayed in triplicate and the average F SD values are reported 894 MOLECULAR THERAPY Vol 10, No 5, November 2004 Copyright C The American Society of Gene Therapy doi:10.1016/j.ymthe.2004.08.006 cofactor to accelerate activation of factor X by factor IXa in the presence of calcium and phospholipids Factor Xa then hydrolyzes a chromogenic substrate and the intensity of the resulting color, which is proportional to factor VIII activity, is determined Using this two-stage assay, we detected 93 F 13 mIU/106 cells/24 h of factor VIII activity in the conditioned medium from the MSGV-sfVIIIDBIRES-EGFP-transduced NIH3T3 cells, whereas we detected no factor VIII activity in the culture supernatant from the nontransduced cells (Fig 2B) We interpreted these results to indicate proper posttranslational processing and secretion of biologically active sfVIIIDB-encoded factor VIII expressed in heterologous cells Sustained Phenotypic Correction of Hemophilia A Mice: MSGV-sfVIIIDB-IRES-EGFP-Directed Expression of Therapeutic Levels of Bioengineered Factor VIII in the Hematopoietic System We transplanted 15 6- to 8-week-old immunocompromised hemophilic E-16 / /B7-2 / mice with or 3.5  106 transduced and sorted BM cells following sublethal (550 cGy total body) g-irradiation We drew peripheral blood from the retro-orbital plexus at regular intervals and analyzed the nucleated cells for EGFP expression by flow cytometry We observed sustained expression of EGFP in all recipients, indicating maintenance of sfVIIIDB-IRES-EGFP transcription Despite the reduced dose conditioning regimen, we observed a high percentage of EGFP-expressing cells in the peripheral blood of most of the transplanted mice (16 F 13% EGFP-positive nucleated cells at 16 weeks posttransplantation; n = 12) as determined by quantitative flow cytometric analysis (Fig 3A) We believe the very low percentage of EGFPexpressing cells in some recipients (e.g., mouse 18) to be due to technical difficulties during intravenous injection We sacrificed three mice (Nos 2, 3, and 4) 15 weeks posttransplantation and transplanted BM collected from two of them (mice and 4) into secondary sublethally irradiated E-16 / /B7-2 / recipients We followed the remaining primary recipients for 20–22 weeks at which time we clipped their tails for coagulation analysis using a stringent survival assay Notably, all transplanted E-16 / /B7-2 / animals exhibited clot formation and survived tail clipping, indicating correction of their hemophilic phenotype In sharp contrast, none of the control untreated hemophilic E-16 / /B7-2 / mice survived tail clipping, dying from exsanguination between and 12 h We detected up to 505 F 42 mIU/ml factor VIII activity in the plasma of the recipient mice by functional COATEST assay at the time tail clipping was performed (Table 1) For comparison purposes, we determined that normal murine plasma contained 680 F 140 mIU/ml factor VIII activity equivalents Interestingly, even the BM recipients with factor VIII levels below the sensitivity of the COATEST assay (mice and 18; b10 mIU/ml factor VIII) survived tail clipping We subse- MOLECULAR THERAPY Vol 10, No 5, November 2004 Copyright C The American Society of Gene Therapy ARTICLE quently sacrificed the mice and analyzed their mononuclear peripheral blood, BM, and spleen cells for EGFP expression by flow cytometry (Table 1), which confirmed the presence of EGFP-positive cells in all of the hematopoietic tissue samples that were examined Transplantation of MSGV-sfVIIIDB-IRES-EGFP-Transduced Primary BM into Secondary Recipients Corrects Hemophilia A To determine whether transduction of HSCs was responsible for the sustained presence of factor VIII in the plasma of the engrafted mice, four sublethally irradiated (550 cGy) E-16 / /B7-2 / mice received BM cells obtained from two primary recipients (mice and 4) sacrificed 15 weeks after transplantation (mouse 2-1 received 1.8  107 cells from donor and mice 2–2 and 4–1 and 4–2 each received  107 cells from donor or 4, respectively) We drew peripheral blood samples periodically from the retro-orbital plexus of these mice and analyzed the nucleated cells for expression of EGFP All four secondary recipients showed persistent expression of EGFP in their peripheral blood mononuclear cells for up to 19 weeks (11 F 4% EGFP positive; n = 4), at which time we clipped their tails (Fig 3B) Although two of these mice had plasma factor VIII levels below the sensitivity of the COATEST assay (b10 mIU/ml), all of the mice exhibited clot formation and survived tail clipping, indicating phenotypic correction of their hemophilia (Table 1) EGFP-expressing cells were detected in the BM and spleens of these secondary recipients at the time of sacrifice (Table 1) Detection and Expression of MSGV-sfVIIIDB-IRES-EGFP Vector Sequences in Spleen Cells of Primary and Secondary Transplant Recipients We analyzed spleen cells from selected primary (mice and 19) and secondary (mice 4–1 and 4–2) transplant recipients for the presence of integrated MSGV-sfVIIIDBIRES-EGFP vector sequences and expression of sfVIIIDBIRES-EGFP transcripts by PCR and RT-PCR, respectively The primers used for PCR were designed such that they would specifically recognize sequences within the region of the sfVIIIDB transgene that had been previously conservatively mutagenized [15] and which therefore differed significantly from the murine factor VIII genomic sequences that remained in the E-16 / /B72 / mice The PCR assay resulted in amplification of a diagnostic 425-bp sfVIIIDB fragment in spleen DNA from all four transplant recipients but not in spleen DNA from control E-16 / /B7-2 / mice (Fig 4A) For the RT-PCR analysis, we used EGFP-specific primers to demonstrate the presence of a 249-bp band in spleen RNA from the four selected primary and secondary transplant recipients following reverse transcription that was not present in similarly treated spleen RNA from control E-16 / /B72 / mice (Fig 4B) These results provided formal proof 895 ARTICLE doi:10.1016/j.ymthe.2004.08.006 FIG Reconstitution kinetics of the peripheral blood of reduced dose conditioned MSGV-sfVIIIDB-IRES-EGFP BM transplant recipients with EGFP-positive cells Shown is the percentage of EGFP-positive nucleated peripheral blood cells in (A) primary and (B) secondary E-16 / /B7-2 / recipients that received MSGVsfVIIIDB-IRES-EGFP-transduced BM cells as determined by quantitative flow cytometric analysis at various time points posttransplantation See text and Table for details of long-term in vivo expression of the sfVIIIDB-IRES-EGFP transcriptional unit—for up to 21 weeks in primary recipients (mouse 19) and 19 weeks in secondary recipients (mice 4–1 and 4–2)—following serial transplantation of E-16 / /B7-2 / mice with MSGV-sfVIIIDBIRES-EGFP-transduced BM cells DISCUSSION In this study we evaluated the potential utility of longterm hematopoietic repopulating cells in murine BM (i.e., HSCs) as a target for hemophilia A gene therapy utilizing an oncoretroviral vector [1,5] We showed sustained production of therapeutic levels of a bioengineered factor VIII protein in minimally myeloablated E-16 / /B7-2 / BM transplant recipients, which resulted in correction of 896 the hemophilic phenotype in 100% of the mice treated It is particularly noteworthy that plasma factor VIII activity in some mice with correspondingly low levels of BM engraftment was below the detection level of the functional assay we used (COATEST), in agreement with clinical observations that nominal elevation of factor VIII plasma levels can result in improvement of the bleeding tendency and convert severe hemophilia to a moderate deficiency [1–4] Previous studies involving mice transplanted with BM cells transduced with human B-domain-deleted factor VIII-encoding oncoretroviruses failed to demonstrate functional factor VIII protein in the plasma [9,10] Hoeben and colleagues [9] were unable to detect any factor VIII transcripts or protein in vivo following transplantation of 2.5  106 preselected BM cells into lethally MOLECULAR THERAPY Vol 10, No 5, November 2004 Copyright C The American Society of Gene Therapy ARTICLE doi:10.1016/j.ymthe.2004.08.006 TABLE 1: Phenotypic correction of hemophilia A mice Mouse Peripheral blood No cells transplanted % EGFP-positive cellsa Weeks posttransplant BM Spleen Factor VIIIb (mIU/ml) Survivalc Primary recipients 10 11 16 17 18 19 20 21  106  106  106  106  106  106  106  106  106 3.5  106 3.5  106 3.5  106 3.5  106 3.5  106 3.5  106 15 15 15 21 22 21 21 21 22 20 20 20 21 21 21 12.5 7.2 19.3 6.5 32.2 10.9 7.8 16.3 9.2 5.4 5.5 2.6 32.6 8.4 13.4 9.6 14.5 39 4.7 22.4 7.3 14.9 20.4 7.4 7.8 ND 1.3 29 8.9 11.3 NDd ND ND 4.9 ND 5.7 5.3 ND ND 5.4 3.5 1.6 ND ND ND ND ND ND 102 F 16 85 F 19 b10 141 F 63 259 F 75 297 F 70 109 F 19 39 F 13 b10 505 F 42 125 F 28 96 F 28 ND ND ND + + + + + + + + + + + + Secondary recipients 2–1 2–2 4–1 4–2 1.8  107  107  107  107 19 19 19 19 7.2 8.1 10.9 16.5 2.3 2.3 6.1 3.2 3.4 2.5 3.5 b10 b10 190 F 30 65 F 25 + + + + b10 680 F 140 0/4 4/4 Controls E-16–/–/B7-2–/– C57BL/6 a b c Percentage of nucleated cells expressing EGFP in peripheral blood, BM, and spleen determined by flow cytometric analysis at time of tail clipping or sacrifice Mean F SD (from triplicate determinations) All MSGV-sfVIIIDB-IRES-EGFP BM transplant recipients evaluated survived tail clipping (+) as did C57BL/6 mice, whereas naRve E-16–/–/B7-2–/– mice died within 2–12 h irradiated wild-type mice It was suggested that perhaps irreversible inactivation of the vector LTR enhancer/ promoter occurred concomitant with differentiation of the hematopoietic stem/progenitor cells We previously demonstrated low levels of factor VIII mRNA in the BM following transplantation of 1–2  106 nonselected BM cells into lethally irradiated factor VIII (exon 17) knockout mice, but plasma levels of factor VIII were below detection [10] In that study, it was found that 30–50% of the transplanted mice became tolerized to factor VIII, indicating that very low levels of factor VIII protein may have been synthesized by antigen-presenting cells [10] These results had suggested that while HSCs are an excellent target for induction of tolerance, they may not be suitable for production of therapeutic levels of factor VIII More recent studies utilizing lentiviral vectors encoding a factor VIII transgene demonstrated secretion of high levels of factor VIII in vitro by various hematopoietic cell lines or primary hematopoietic cells [19,23,24] These studies argued that inability to detect oncoretroviral vector-directed factor VIII in vivo in the earlier BM transplant studies was not due to an absolute block in biosynthesis or secretion of factor VIII in hematopoietic cells MOLECULAR THERAPY Vol 10, No 5, November 2004 Copyright C The American Society of Gene Therapy Based on the above information, we were interested in reevaluating the feasibility of HSC-directed hemophilia A gene therapy using a modified oncoretroviral vector The MSGV-sfVIIIDB-IRES-EGFP vector employed in this study is a derivative of the MSCV oncoretroviral vector [11], which is known to attain high-level in vivo expression of transgenes in a variety of hematopoietic cells of murine and human origin [12–14] The MSCV LTR contains modifications that may render it more resistant to transcriptional silencing mechanisms operating in primitive hematopoietic precursors [5,25] This salient feature of MSCV distinguishes it from the oncoretroviral vectors used in the earlier studies [9,10] and may have played a central role in therapeutic plasma levels of factor VIII being achieved in the present work Indeed, transcriptional silencing and extinction of factor VIII transgene expression in vivo appear to have been the reason for the failure of different hemophilia A gene therapy approaches utilizing other oncoretroviral vector platforms [26,27] Clearly, ex vivo preselection of the transduced BM cells helped facilitate maintenance of factor VIII transgene expression in vivo [28] However, as mentioned above, Hoeben and colleagues [9] also transplanted preselected 897 ARTICLE FIG Detection of MSGV-sfVIIIDB-IRES-EGFP vector sequences and expression in primary and secondary BM transplant recipients (A) The sfVIIIDB transgene was detected as a 425-bp fragment by PCR in genomic DNA prepared from spleen cells obtained from selected primary (mice and 19) and secondary (mice 4–1 and 4–2) E-16 / /B7-2 / recipients of MSGVsfVIIIDB-IRES-EGFP-transduced BM cells MSGV-sfVIIIDB-IRES-EGFP vector DNA was used as a positive control Genomic DNA prepared from spleen cells obtained from a naRve E-16 / /B7-2 / mouse was used as a negative control A 100-bp ladder molecular weight marker was included for sizing of DNA fragments (B) The sfVIIIDB-IRES-EGFP transcriptional unit was detected by RTPCR analysis (+) of total RNA prepared from spleen cells obtained from the mice described in A Primers were designed to amplify a 249-bp fragment of the EGFP gene PCR analysis of RNA samples without ( ) addition of reverse transcriptase demonstrated lack of genomic DNA amplification Total RNA prepared from spleen cells obtained from a naRve E-16 / /B7-2 / mouse was used as a negative control A 100-bp ladder molecular weight marker was included for sizing of DNA fragments transduced BM cells in their series of experiments to no avail Other aspects of the experimental design most likely contributed to the success of the current investigations While B-domain-deleted factor VIII transgenes have universally been used for oncoretroviral-mediated gene therapy strategies because of size constraints and decreased expression associated with the full-length factor VIII gene [29], incorporation of the MFG vector env splice acceptor site into the oncoretroviral vector backbone undoubtedly results in higher factor VIII mRNA levels due to enhanced splicing [15–17] Nevertheless, this enhancement is apparently insufficient per se since the factor VIII oncoretroviral vector that we previously employed to express the factor VIII gene also contained a 5V intron [10] To achieve more efficient synthesis of factor VIII, the sfVIIIDB open reading frame was provided with a Kozak consensus translation initiation signal In addition, the sfVIIIDB factor VIII transgene contains two point mutations (L303E/F309S) in the A1 domain that were shown previously to increase the 898 doi:10.1016/j.ymthe.2004.08.006 secretion of factor VIII about threefold in heterologous cells by disrupting its interaction with the protein chaperone BiP, thus facilitating its release from the endoplasmic reticulum to the Golgi apparatus [18] In support of this notion, another bioengineered form of factor VIII having the F309S point mutation has recently been reported to be secreted more efficiently from heterologous cells than the B-domain-deleted factor VIII protein [30] Finally, in the above-referenced lentiviral gene transfer studies of Kootstra and colleagues [19], only low levels of factor VIII could be transiently detected in plasma of immunocompetent hemophilia A mice that received transduced BM cells, which was reportedly due to the development of factor VIII-neutralizing antibodies that led to elimination of the gene-modified cells Induction of a factor VIII-specific immune response in immunocompetent factor VIII-naRve mice mimics the undesirable appearance of inhibitory antibodies in hemophilia A patients undergoing replacement therapy [1–4] Therefore, to circumvent this possibility, which would have precluded the opportunity to evaluate whether there were inherent limitations preventing production of therapeutic plasma levels of factor VIII by HSCtargeted gene transfer, we used factor VIII-deficient mice that were also deficient in the T cell costimulatory ligand B7-2/CD86 [20] It had previously been shown that these mice not mount T cell or antibody responses to factor VIII even following repeated intravenous infusions of factor VIII immunogen [20] The fact that factor VIII and EGFP expression were maintained for at least months in the primary transplant recipients suggested that an HSC subpopulation had been transduced Confirmatory data were provided by the secondary BM transplants, which showed longterm (almost months) expression of the sfVIIIDB-IRESEGFP cassette in peripheral blood, BM, and spleen cells It is known that in addition to HSCs forming all blood lineages, BM also contains mesenchymal stem cells [31] Since we did not enrich for HSCs, BM stromal cellsincluding BM-resident mesenchymal stem cells-could also have been transduced However, given the lower frequencies of mesenchymal stem cells in BM harvests (2– per 106 total nucleated cells versus 1–10 per 105 for HSCs) [32] and the difficulty in obtaining mesenchymal stem cells from murine BM preparations [33], we think it unlikely that these target cell types would have contributed meaningfully to the durable in vivo transgenic human factor VIII production that we observed It has been shown in both small and large animal models that introduction of transgenes via HSCs can induce immunological tolerance to the vector-encoded neoantigen [10,34–39] Of central relevance in this context, we were able to induce immune tolerance to human factor VIII in 30–50% of hemophilic mice transplanted with gene-modified BM cells expressing Bdomain-deleted human factor VIII [10] However, the MOLECULAR THERAPY Vol 10, No 5, November 2004 Copyright C The American Society of Gene Therapy doi:10.1016/j.ymthe.2004.08.006 animals in that study were preconditioned by lethal (900 cGy total body) g-irradiation While fully myeloablative preconditioning regimens allow high-level donor chimerism following BM transplantation, they are undesirable for treatment of hemophilia patients Previous studies suggested that clinically relevant levels of engraftment by transplanted BM cells could be achieved at approximately 500 cGy total body g-irradiation [36,40] After conditioning using a sublethal dose of girradiation (550 cGy), we demonstrated here that adequate BM engraftment could be achieved in the absence of overt radiation toxicity, resulting in plasma factor VIII levels that corrected the hemophilic phenotype To induce tolerance to a transgene product delivered via BM transplantation, a certain degree of hematopoietic microchimerism resulting in a minimal threshold of transgene expression in donor antigenpresenting cells appears to be sufficient [35–38] Therefore, in our future studies in immunocompetent hemophilia A mice, efforts will be focused on optimizing tolerance induction to transgenic factor VIII with various nonmyeloablative conditioning regimens while striving to retain therapeutic factor VIII plasma levels In view of the recent cases of leukemia-like syndrome that developed following gene therapy of X-linked severe combined immunodeficiency patients [7,41], a major concern in clinical HSC gene transfer studies involving oncoretroviral vectors is their biosafety profile Although it is not expected that the risk of insertional leukemogenesis in hemophilia A patients transplanted with genemodified HSCs would be similarly high [42], it would be beneficial if the safety features that have been introduced into current-generation lentiviral vectors—self-inactivating LTR format and inclusion of enhancer-blocking insulators—could be incorporated into future factor VIII oncoretroviral vectors [5,43,44] In summary, the findings reported open a new avenue in hemophilia A gene therapy modeling Further BM transplant studies using immunocompetent factor VIIIdeficient mice are planned to confirm and extend the data, especially the potential to induce tolerance to MSGV-sfVIIIDB-IRES-EGFP-expressed factor VIII in nonmyeloablated recipients, before prospective clinical correction of the hemophilic phenotype might be envisaged using this approach MATERIALS AND METHODS Factor VIII-deficient mice Six- to 8-week-old E-16 / /B7-2 / immunocompromised hemophilic male or female factor VIII (exon 16) knockout mice also deficient for the T cell costimulatory ligand B7-2/CD86 were used as BM transplant donors and recipients [20] The E-16 / /B7-2 / doubleknockout mice were derived previously by cross-breeding of E-16 / mice with B7-2 / knockout mice [20] Breeding colonies of E-16 / /B7-2 / mice were maintained by breeding of hemizygous affected males and homozygous affected females These mice exhibit factor VIII activity of b1% of normal levels by analysis with the COATEST assay (described MOLECULAR THERAPY Vol 10, No 5, November 2004 Copyright C The American Society of Gene Therapy ARTICLE below) All animal procedures were carried out in accordance with Institutional Animal Care and Use Committee guidelines Factor VIII oncoretroviral vector construction and production of vector conditioned medium The oncoretroviral vector used in all experiments was MSGV-sfVIIIDB-IRES-EGFP MSGV (MSCV-based splice-gag vector) is a derivative of the MSCV [11] and contains an extended gag region and env splice site It was generated from MSCV-based MINV vector backbone [45] by substituting a 756-bp SpeI/XhoI fragment with an 1143-bp SpeI/XhoI fragment from the MFG-based vector SFGtcLuc+ITE4 [46] (a gift from Dr Richard Mulligan, Harvard Medical School, Boston, MA, USA) and by replacing a 1955-bp XhoI/BamHI fragment containing a pgk-IRES-neo cassette with a 47-bp XhoI/BamHI polylinker containing unique XhoI, EcoRI, SalI, SacII, and BamHI sites The factor VIII transgene employed was derived from a B-domain-deleted (minus nucleotides 2335–4974) factor VIII cDNA F8(3Vmut) in which the 3V half (655 bp) of a 1.2-kb putative inhibitory sequence had been mutated conservatively in a previous study [15] The F8(3Vmut) cDNA was further modified to generate a secretion-enhanced factor VIII (sfVIIIDB) mutant as follows: the translational initiation site was incorporated within a Kozak consensus sequence and two point mutations (L303E/F309S) were introduced into the A1 domain by oligonucleotide overlap extension PCR as described previously [15,18] A 4663-bp XhoI fragment containing the sfVIIIDB cDNA was excised from SuperF8 (R.A.M., unpublished) and subcloned into the XhoI/SalI sites present in the MSGV polylinker Finally, a 1415-bp EcoRI/XhoI fragment containing an EGFP reporter gene linked to an encephalomyocarditis virus internal ribosome entry site cassette from pBSP-IRES-EGFP (A.R and R.G.H., unpublished) was blunt-end-ligated into a SalI site present at the 3Vend of the sfVIIIDB sequences to create MSGV-sfVIIIDB-IRES-EGFP The Phoenix-Eco packaging cell line (ATCC No SD 3444; American Type Culture Collection, Manassas, VA, USA) was grown in Dulbecco’s modified Eagle’s medium (Invitrogen Corp., Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Cambrex Bio Science Walkersville, Inc., Walkersville, MD, USA), l-glutamine (2 mM; Invitrogen Corp.), penicillin (50 IU/ml), and streptomycin (50 Ag/ml; Invitrogen Corp.) at 378C and 5% CO2 Phoenix-Eco cells were transiently transfected with MSGV-sfVIIIDB-IRES-EGFP by calcium phosphate coprecipitation [47] and vector conditioned medium was collected after 72 h, centrifuged at 2000g to remove cellular debris, and filtered through a 0.45-Am-pore-size filter (Nalgene) before being aliquoted and frozen at 808C for future transductions To determine the titer of MSGV-sfVIIIDBIRES-EGFP stocks, an aliquot of Phoenix-Eco vector conditioned medium was thawed and serial dilutions were added in the presence of Ag/ml Polybrene (hexadimethrine bromide; Sigma, St Louis, MO, USA) to  105 NIH3T3 cells (ATCC No CRL-1658) that had been seeded in six-well plates h earlier Fresh medium was added after h of transduction, and 72 h later the relative end-point vector titer—approximately 0.8–1.0  106 transducing units/ml (TU/ml)—was determined by flow cytometric analysis on a BD LSR benchtop analyzer (BD Biosciences, San Jose, CA, USA) by multiplying the percentage of EGFP-positive cells by the number of cells plated at the time of transduction (2  105) and the dilution factor BM cell transduction Total BM cells were obtained from 6- to 8-week-old E-16 / /B7-2 / mice by flushing the hind limbs with phosphate-buffered saline (PBS) containing 2% FBS Red blood cells (RBCs) were lysed by incubating total BM cells with 20 ml Puregene RBC lysis solution (Gentra Systems, Inc., Minneapolis, MN, USA) for 10 at room temperature followed by centrifugation The nucleated cell fraction was then transferred to plates coated with full-length human fibronectin (BD Biosciences) and cultured in Iscove’s modified Dulbecco’s medium (Cambrex Bio Science Walkersville, Inc.) supplemented with 10% heat-inactivated FBS and 5% conditioned medium from X630-rIL-3 cells (a source of recombinant murine IL-3; a gift from Dr Fritz Melchers, University of Basel, Basel, Switzerland) [48], 5% conditioned medium from Sp2/mIL-6 cells (a source of recombinant murine IL-6 [49]), and 10% conditioned medium from Chinese hamster ovary cells producing soluble murine stem cell factor [12] After 48 h of prestimulation, the BM cells were transduced 899 ARTICLE for consecutive days (4 h each day) by incubation with MSGV-sfVIIIDBIRES-EGFP vector conditioned medium and Ag/ml Polybrene supplemented with the same growth factors as used for prestimulation Transduction efficiency ranged from 17 to 32% The cells expressing EGFP were sorted 48 h after the final transduction and immediately injected intravenously into sublethally irradiated recipient mice (described below) Cell sorting was performed on a triple-laser FACSVantage SE instrument with digital electronic option (BD Biosciences) The EGFP fluorescent signal was detected in the FL1 channel through a 530/30 bandpass filter after excitation at 488 nm with an Innova 70C-Spectrum mixed argonkrypton ion laser (Coherent, Inc., Santa Clara, CA, USA) Viable cells were gated by a combination of forward and orthogonal light scatter, and data were acquired and analyzed using FACSDiva software (BD Biosciences) BM transplantation and assessment of phenotypic correction Six- to 8week-old E-16 / /B7-2 / mice were injected intravenously via tail vein with or 3.5  106 transduced and sorted BM cells (mice to received 1.8  106 EGFP-positive cells, mice to 11 received 1.6  106 EGFPpositive cells, and mice 16 to 21 received 2.9  106 EGFP-positive cells) Immediately before transplantation, the recipients received a sublethal dose of 550 cGy total body g-irradiation from a 137Cs source Three of these mice (2, 3, and 4) were sacrificed at 15 weeks posttransplantation and BM cells from two of them (mice and 4) were injected into two sublethally irradiated secondary transplant E-16 / /B7-2 / recipients each (mouse 2–1 received 1.8  107 unsorted BM cells and mice 2–2, 4–1, and 4–2 each received  107 cells) The remainder of the primary recipients and the secondary recipients were evaluated for phenotypic correction by tail clipping [50] at 20 to 22 weeks after primary transplant and 19 weeks after secondary transplant, respectively Briefly, under isoflurane anesthesia the tail was pulled through a hole with a 1.58-mm diameter until snug to ensure cuts of identical cross-sectional area between different mice The cut was made approximately 2.75–3.5 cm from the tail tip Before tail clipping was performed, blood was obtained from the retroorbital plexus in 1/10 volume of 0.1 M sodium citrate (2.94%) and centrifuged at 2000g for 10 min, the plasma was frozen on dry ice immediately and then stored at 808C for future analysis The RBCs were then lysed and the remaining nucleated cells were analyzed for expression of EGFP by flow cytometry Single-cell suspensions of spleens and BM cells from some of the recipient mice were prepared and, following lysis of the RBCs, the nucleated cells were analyzed for expression of EGFP by flow cytometry Analysis of factor VIII activity Factor VIII activity was determined by a chromogenic functional assay (COATEST VIII:C/4; DiaPharma Group, Inc., West Chester, OH) according to the manufacturer’s instructions All samples were assayed in triplicate and the means calculated Reconstituted, normal pooled human plasma (Calibration plasma; DiaPharma Group, Inc., West Chester, OH, USA), which has 1000 mIU or 100% activity, equivalent to 200 ng/ml, was used to generate the standard curve NIH3T3 cells were transduced with MSGV-sfVIIIDB-IRES-EGFP vector particles at a concentration of  106 TU per  105 cells for h in the presence of Ag/ml Polybrene and then sorted for EGFP-expressing cells 72 h later Conditioned medium was collected from transduced and nontransduced NIH3T3 cells for determination of factor VIII activity For determination of in vivo sfVIIIDB expression, factor VIII activity was assayed in citrated murine plasma Frozen plasma samples were thawed in a 378C water bath and immediately assayed by COATEST The positive and negative controls were pooled plasma from 10 C57BL/6 and E-16 / /B7-2 / mice, respectively Western blot analysis Conditioned medium was collected from transduced and nontransduced NIH3T3 cells and diluted 1.5:1 with Hepes lysis buffer (20 mM Hepes, 0.5 M NaCl, mM EDTA, 0.25% Triton X-100, mM EGTA) supplemented with a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA) and 0.1 mM PMSF The cultured cells were then lysed and precleared by overnight incubation with protein G- 900 doi:10.1016/j.ymthe.2004.08.006 Sepharose beads (Protein-G Immunoprecipitation Kit; Sigma) and immunoprecipitated with two light-chain-specific monoclonal antibodies (ESH2 and ESH8; American Diagnostica, Inc., Stamford, CT, USA) The immunoprecipitated samples were electrophoresed on a 4–12% Bis-Tris NuPAGE gel (Invitrogen Corp.) under reducing conditions in parallel with 20 ng of recombinant human factor VIII as control (American Diagnostica, Inc.) and transferred to an Immobilon-P PVDF membrane (Sigma) The membrane was blocked for h at 258C with 5% nonfat milk in PBS with 0.05% Tween 20 and then blotted with an affinity-purified polyclonal sheep anti-human factor VIII antibody (1:1000, SAF8C-AP; Enzyme Research Laboratories, South Bend, IN, USA) for h at room temperature To visualize the factor VIII-specific antibody, an alkaline phosphatase-conjugated rabbit anti-sheep secondary antibody (1:20,000; Pierce Biotechnology, Rockford, IL, USA) was used Chemifluorescence detection was performed with ECF substrate (Amersham Biosciences Corp., Piscataway, NJ, USA) and the blot scanned on a Storm 860 PhosphorImager using ImageQuant software (Amersham Biosciences Corp.) Southern and Northern blot analyses Southern and Northern blot analyses were carried out as described [44,51] In brief, genomic DNA (10 Ag) from each sample was digested with SacI, separated on 1% agarose gels, and transferred to Hybond-N+ membranes (Amersham Biosciences Corp.) in 5 SSC (1 SSC is 0.15 M NaCl plus 0.015 M sodium citrate) Total cellular RNA (20 Ag) was separated on 1.2% agarose-formaldehyde gels and subsequently transferred to Hybond-N+ membranes in 5 SSC Membranes were fixed by exposure to UV light and hybridized with 32Plabeled randomly primed probes having specific activities of 1–5  108 dpm/Ag A 0.8-kb fragment of pEGFP-1 (BD Biosciences Clontech, Palo Alto, CA, USA) was used as an EGFP-specific probe; a 1.3-kb EcoRI-AccI fragment of pCDj-989 was used to detect the endogenous murine bcl2 gene [52], and a 1.8-kb fragment containing the human h-actin cDNA (BD Biosciences Clontech) was used as a probe for murine h-actin transcripts Hybridizations were performed overnight in 4 SSC, 20% (w/v) dextran sulfate, 5 Denhardt’s solution, 0.05% SDS, and 100 Ag/ml salmon sperm DNA at 428C Following hybridization, membranes were washed twice in 1 SSC-1% SDS for 30 and once in 0.1 SSC-0.1% SDS for 30 at 558C and then exposed to storage phosphor screens Digital images were acquired using a Storm 860 PhosphorImager and radioactivity was quantitated using ImageQuant software PCR and RT-PCR analyses To detect integrated vector sequences in spleen genomic DNA samples, PCR was performed using a pair of sfVIIIDB-specific primers: sF8-5V (5V-GCCAGAAACAGTTCAACG-3V) and sF8-3V (5V-CACCATAATGTTGTCCTC-3V) These primers amplify a 425-bp region within the 3V half of the 1.2-kb inhibitory sequence that is conservatively mutated in sfVIIIDB and not detect murine factor VIII sequences Genomic DNA (1 Ag) was used in a 50-Al PCR containing Al of 10 buffer [100 mM Tris-HCl, pH 8.85, 250 mM KCl, 50 mM (NH4)2SO4, 20 mM MgSO4], 125 AM each dNTP, 30 pmol forward primer (sF8-5V), 30 pmol reverse primer (sF8-3V), and 2.5 U of Taq-Pwo DNA polymerase (Roche Diagnostics) Following an initial denaturation step at 948C for min, the thermocycle profile, repeated 40 times, consisted of a step cycle of 948C for 15 s, 508C for 30 s, and 728C for min, with a final 7-min elongation step at 728C The 425-bp PCR-amplified products were then separated on a 1.6% agarose gel MSGV-sfVIIIDB-IRES-EGFP plasmid DNA was used as a positive control and 100 ng was amplified under the same conditions Spleen samples obtained from the transplanted mice were homogenized and total RNA was isolated using Trizol reagent (Invitrogen Corp.) For the detection of vector RNA, RT-PCR was performed using a pair of EGFP-specific primers: GFP-5V (5V-CACAAGTTCAGCGTGTCC-3V) and GFP-3V (5V-CTTGTAGTTGCCGTCGTC-3V) Total RNA (2 Ag) was mixed with the GFP-3V primer (20 pmol) in a 9-Al volume, heated to 658C for 10 min, and chilled on ice for The reaction volume was then increased to 20 Al and the reverse transcription was performed in the presence of Al of 5 buffer (50 mM Tris-HCl, 200 mM KCl, 30 mM MgCl2, 50 mM dithioerythritol, pH 8.3), 125 AM each dNTP, and 40 units of M-MLV reverse transcriptase (Roche Diagnostics) Following h MOLECULAR THERAPY Vol 10, No 5, November 2004 Copyright C The American Society of Gene Therapy doi:10.1016/j.ymthe.2004.08.006 incubation at 378C, the reaction was stopped by 10 heat inactivation at 658C The cDNA (5 Al reaction mixture) was then amplified in a 50-Al PCR containing Al of 10 buffer [100 mM TrisHCl, pH 8.85, 250 mM KCl, 50 mM (NH4)2SO4, 20 mM MgSO4], 125 AM each dNTP, 30 pmol forward primer (GFP-5V), 30 pmol reverse primer (GFP-3V), and 2.5 U of Taq-Pwo DNA polymerase (Roche Diagnostics) Following an initial denaturation step at 948C for min, the thermocycle profile, repeated 35 times, consisted of a step cycle of 948C for 15 s, 658C for 30 s, and 728C for 30 s, with a final 7-min elongation step at 728C The 249-bp PCR-amplified products were then separated on a 1% agarose gel ACKNOWLEDGMENTS This work was supported in part by National Institutes of Health Grants HL65519 and HL66305 (to R.G.H.) 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involve the 5Vor the 3Vregion of the bcl-2 gene Proc Natl Acad Sci USA 84: 1329 – 1331 MOLECULAR THERAPY Vol 10, No 5, November 2004 Copyright C The American Society of Gene Therapy ... identified factor VIII cross-reactive material with an antifactor VIII polyclonal antibody (SAF8C-AP) We used recombinant full-length human factor VIII, comprising primarily a heavy chain migrating at... at 200 kDa and a light chain migrating at 80 kDa, as a positive control Both chains of recombinant human factor VIII were detected with the SAF8C-AP anti -factor VIII polyclonal antibody although... phosphatase-conjugated secondary antibody, and ECF chemifluorescence detection reagent SC, single chain; HC, heavy chain; LC, light chain The heavy and light chains of recombinant human factor VIII