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[.]

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,*

1 Department of Anatomy and Cell Biology and 4 Flow Cytometry Core Facility, The George Washington University Medical Center, Washington, DC 20037, USA 2 Graduate Genetics Program,

The George Washington University, Washington, DC 20052, USA

3 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 3 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 1 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

plasma-derived or recombinant factor VIII protein Very high

cost and unpredictable shortages of recombinant factor

VIII and risk of transmission of certain blood-borne

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

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

oncoretrovi-ral vectors are widely used for HSC gene transfer because

their stable chromosomal integration provides the

oppor-tunity 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

oncoretrovi-ral-mediated gene delivery approaches were unsuccessful

[9,10]

In this paper we describe the construction of

MSGV-sfVIIIDB-IRES-EGFP, a murine stem cell virus

(MSCV)-based oncoretroviral vector [11] carrying a

B-domain-deleted factor VIII cDNA (sfVIIIDB) bioengineered for

enhanced secretion The MSCV vector has been

demon-strated 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

mye-loablated primary and secondary recipients with

MSGV-sfVIIIDB-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

helper-free ecotropic MSGV-sfVIIIDB-IRES-EGFP vector particles

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 secre-tion of sfVIIIDB-encoded protein from transduced NIH3T3 cells by Western blot analysis (Fig 2A) We harvested culture supernatants and extracts from trans-duced EGFP-sorted and control NIH3T3 cells and per-formed immunoprecipitations with two factor VIII light-chain-specific monoclonal antibodies (ESH2 and ESH8) Following polyacrylamide gel electrophoresis and trans-fer to a polyvinylidene difluoride (PVDF) membrane, we identified factor VIII cross-reactive material with an anti-factor 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 anti-body 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-IRES-EGFP-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 char-acteristic 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

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 2 Secretion of functional factor VIII from

MSGV-sfVIIIDB-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

poly-clonal 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/10 6 cells/24 h All samples were

assayed in triplicate and the average F SD values are

reported.

FIG 1 Structure of the MSGV-sfVIIIDB-IRES-EGFP vector

and expression in transduced NIH3T3 cells (A)

Sche-matic 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,

secre-tion-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

complete-ness of DNA digestion (C) Northern blot analysis Total

RNA was extracted from nontransduced and

trans-duced NIH3T3 cells and vector transcripts were

detected by hybridization with an EGFP-specific probe.

The blot was rehybridized with a probe specific for

h-actin sequences to monitor intactness of the RNA.

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

inten-sity of the resulting color, which is proportional to factor

VIII activity, is determined Using this two-stage assay, we

detected 93 F 13 mIU/106cells/24 h of factor VIII activity

in the conditioned medium from the

MSGV-sfVIIIDB-IRES-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

immunocompro-mised hemophilic E-16 / /B7-2 / mice with 2 or 3.5 

106transduced 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

percent-age 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

EGFP-expressing 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 2 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

for-mation 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 2 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

deter-mined 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 8 and 18; b10

mIU/ml factor VIII) survived tail clipping We

subse-quently sacrificed the mice and analyzed their mono-nuclear 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 hema-topoietic 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 respon-sible 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 2 and 4) sacrificed 15 weeks after transplantation (mouse 2-1 received 1.8  107cells from donor 2 and mice 2–2 and 4–1 and 4–2 each received 1  107cells from donor 2 or 4, respectively) We drew peripheral blood samples periodi-cally 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 4 and 19) and secondary (mice 4–1 and 4–2) transplant recipients for the presence of integrated MSGV-IRES-EGFP vector sequences and expression of sfVIIIDB-IRES-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 /

/B7-2 / 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 recipi-ents following reverse transcription that was not present

in similarly treated spleen RNA from control E-16 /

/B7-2 / mice (Fig 4B) These results provided formal proof

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

trans-plantation of E-16 / /B7-2 / mice with

MSGV-sfVIIIDB-IRES-EGFP-transduced BM cells

DISCUSSION

In this study we evaluated the potential utility of

long-term 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

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 func-tional 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 trans-plantation of 2.5  106preselected BM cells into lethally

FIG 3 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 MSGV-sfVIIIDB-IRES-EGFP-transduced BM cells as determined by quantitative flow cytometric analysis at various time points posttransplantation See text and Table 1

for details.

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  106nonselected BM

cells into lethally irradiated factor VIII (exon 17)

knock-out 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

hema-topoietic cell lines or primary hemahema-topoietic 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

Based on the above information, we were interested

in reevaluating the feasibility of HSC-directed hemo-philia 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 oper-ating 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 utiliz-ing 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

TABLE 1: Phenotypic correction of hemophilia A mice

Mouse

No cells transplanted

Weeks posttransplant

% EGFP-positive cellsa Factor VIIIb

Primary recipients

Secondary recipients

Controls

E-16 –/– /B7-2 –/–

a Percentage of nucleated cells expressing EGFP in peripheral blood, BM, and spleen determined by flow cytometric analysis at time of tail clipping or sacrifice.

b Mean F SD (from triplicate determinations).

c 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.

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]

Never-theless, 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

initia-tion signal In addiinitia-tion, the sfVIIIDB factor VIII transgene

contains two point mutations (L303E/F309S) in the A1

domain that were shown previously to increase the

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 hemo-philia 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 HSC-targeted 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 do 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 4 months in the primary trans-plant recipients suggested that an HSC subpopulation had been transduced Confirmatory data were provided

by the secondary BM transplants, which showed long-term (almost 5 months) expression of the sfVIIIDB-IRES-EGFP 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 cells-including BM-resident mesenchymal stem cells-could also have been transduced However, given the lower frequencies of mesenchymal stem cells in BM harvests (2–

5 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 con-tributed 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 B-domain-deleted human factor VIII [10] However, the

FIG 4 Detection of MSGV-sfVIIIDB-IRES-EGFP vector sequences and

expres-sion 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 4 and 19)

and secondary (mice 4–1 and 4–2) E-16 / /B7-2 / recipients of

MSGV-sfVIIIDB-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

RT-PCR 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.

animals in that study were preconditioned by lethal

(900 cGy total body) g-irradiation While fully

myeloa-blative preconditioning regimens allow high-level donor

chimerism following BM transplantation, they are

undesirable for treatment of hemophilia patients

Pre-vious 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

g-irradiation (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

pheno-type 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

antigen-presenting cells appears to be sufficient [35–38]

There-fore, in our future studies in immunocompetent

hemo-philia 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

leukemo-genesis in hemophilia A patients transplanted with

gene-modified HSCs would be similarly high[42], it would be

beneficial if the safety features that have been introduced

into current-generation lentiviral

vectors—self-inactivat-ing LTR format and inclusion of enhancer-blockvectors—self-inactivat-ing

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

VIII-deficient mice are planned to confirm and extend the

data, especially the potential to induce tolerance to

MSGV-sfVIIIDB-IRES-EGFP-expressed factor VIII in

non-myeloablated recipients, before prospective clinical

cor-rection 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 /

immuno-compromised 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 /

double-knockout 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

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 trans-lational 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) supple-mented 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% CO 2 Phoenix-Eco cells were transiently transfected with MSGV-sfVIIIDB-IRES-EGFP by calcium phosphate copre-cipitation [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-sfVIIIDB-IRES-EGFP stocks, an aliquot of Phoenix-Eco vector conditioned medium was thawed and serial dilutions were added in the presence of 8 Ag/ml Polybrene (hexadimethrine bromide; Sigma, St Louis, MO, USA) to 2 

10 5 NIH3T3 cells (ATCC No CRL-1658) that had been seeded in six-well plates 8 h earlier Fresh medium was added after 2 h of transduction, and

72 h later the relative end-point vector titer—approximately 0.8–1.0  10 6 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  10 5 ) 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 min at room temperature followed by centrifugation The nucleated cell fraction was then trans-ferred to plates coated with full-length human fibronectin (BD Bioscien-ces) 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

for 3 consecutive days (4 h each day) by incubation with

MSGV-sfVIIIDB-IRES-EGFP vector conditioned medium and 8 Ag/ml Polybrene

supple-mented with the same growth factors as used for prestimulation

Trans-duction 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

argon-krypton 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

8-week-old E-16 /

/B7-2 / mice were injected intravenously via tail vein

with 2 or 3.5  10 6 transduced and sorted BM cells (mice 2 to 4 received

1.8  10 6 EGFP-positive cells, mice 6 to 11 received 1.6  10 6

EGFP-positive cells, and mice 16 to 21 received 2.9  10 6 EGFP-positive cells).

Immediately before transplantation, the recipients received a sublethal

dose of 550 cGy total body g-irradiation from a 137 Cs source Three of

these mice (2, 3, and 4) were sacrificed at 15 weeks posttransplantation

and BM cells from two of them (mice 2 and 4) were injected into two

sublethally irradiated secondary transplant E-16 / /B7-2 / recipients

each (mouse 2–1 received 1.8  107unsorted BM cells and mice 2–2, 4–1,

and 4–2 each received 1  10 7 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

retro-orbital 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

Reconsti-tuted, 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 1  10 6 TU per 2  10 5 cells for 2 h in

the presence of 8 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

trans-duced and nontranstrans-duced NIH3T3 cells and diluted 1.5:1 with Hepes lysis

buffer (20 mM Hepes, 0.5 M NaCl, 1 mM EDTA, 0.25% Triton X-100, 1

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-Sepharose beads (Protein-G Immunoprecipitation Kit; Sigma) and immu-noprecipitated 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 Diagnos-tica, Inc.) and transferred to an Immobilon-P PVDF membrane (Sigma) The membrane was blocked for 1 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 1 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 32 P-labeled randomly primed probes having specific activities of 1–5  10 8 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 min and once in 0.1 SSC-0.1% SDS for 30 min 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 do not detect murine factor VIII sequences Genomic DNA (1 Ag) was used in a 50-Al PCR containing 5 Al of 10 buffer [100 mM Tris-HCl, pH 8.85, 250 mM KCl, 50 mM (NH4) 2 SO 4 ,

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 2 min, the thermocycle profile, repeated 40 times, consisted of a step cycle

of 948C for 15 s, 508C for 30 s, and 728C for 1 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 homo-genized 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 2 min The reaction volume was then increased to 20 Al and the reverse transcription was performed in the presence of 2 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 1 h

incubation at 378C, the reaction was stopped by 10 min heat

inactivation at 658C The cDNA (5 Al reaction mixture) was then

amplified in a 50-Al PCR containing 5 Al of 10 buffer [100 mM

Tris-HCl, pH 8.85, 250 mM KCl, 50 mM (NH4) 2 SO 4 , 20 mM MgSO 4 ], 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 2 min, the

thermo-cycle profile, repeated 35 times, consisted of a step thermo-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.

This work was supported in part by National Institutes of Health Grants

HL65519 and HL66305 (to R.G.H.) Portions of this study were performed by

M.M in partial fulfillment of the requirements for the Ph.D degree in Genetics

from the Institute for Biomedical Sciences, The George Washington University,

Washington, DC.

RECEIVED FOR PUBLICATION JUNE 9, 2004; ACCEPTED AUGUST 4, 2004.

REFERENCES

1 Saenko, E L., Ananyeva, N M., Moayeri, M., Ramezani, A., and Hawley, R G (2003).

Development of improved factor VIII molecules and new gene transfer approaches for

hemophilia A Curr Gene Ther 3: 27 – 41.

2 Hoyer, L W (1994) Hemophilia A N Engl J Med 330: 38 – 47.

3 Bolton-Maggs, P H B., and Pasi, K J (2003) Haemophilias A and B Lancet 361:

1801 – 1809.

4 High, K A (2001) Gene transfer as an approach to treating hemophilia Circ Res 88:

137 – 144.

5 Hawley, R G (2001) Progress toward vector design for hematopoietic stem cell gene

therapy Curr Gene Ther 1: 1 – 17.

6 Heim, D A., and Dunbar, C E (2000) Hematopoietic stem cell gene therapy: towards

clinically significant gene transfer efficiency Immunol Rev 178: 29 – 38.

7 Hacein-Bey-Abina, S., et al (2002) Sustained correction of X-linked severe combined

immunodeficiency by ex vivo gene therapy N Engl J Med 346: 1185 – 1193.

8 Aiuti, A., et al (2002) Correction of ADA-SCID by stem cell gene therapy combined

with nonmyeloablative conditioning Science 296: 2410 – 2413.

9 Hoeben, R C., Einerhand, M P W., BriJt, E., van Ormondt, H., Valerio, D., and van der

Eb, A J (1992) Toward gene therapy in haemophilia A: retrovirus-mediated transfer of

a factor VIII gene into murine haematopoietic progenitor cells Thromb Haemostasis

67: 341 – 345.

10 Evans, G L., and Morgan, R A (1998) Genetic induction of immune tolerance to

human clotting factor VIII in a mouse model for hemophilia A Proc Natl Acad Sci USA

95: 5734 – 5739.

11 Hawley, R G., Lieu, F H., Fong, A Z., and Hawley, T S (1994) Versatile retroviral

vectors for potential use in gene therapy Gene Ther 1: 136 – 138.

12 Hawley, R G., et al (1996) Thrombopoietic potential and serial repopulating ability of

murine hematopoietic stem cells constitutively expressing interleukin 11 Proc Natl.

Acad Sci USA 93: 10297 – 10302.

13 Cheng, L., et al (1998) Sustained gene expression in retrovirally transduced, engrafting

human hematopoietic stem cells and their lympho-myeloid progeny Blood 92: 83 – 92.

14 Dorrell, C., Gan, O I., Pereira, D S., Hawley, R G., and Dick, J E (2000) Expansion of

human cord blood CD34 + CD38 cells in ex vivo culture during retroviral transduction

without a corresponding increase in SCID repopulating cell (SRC) frequency:

dissociation of SRC phenotype and function Blood 95: 102 – 110.

15 Chuah, M K L., Vandendriessche, T., and Morgan, R A (1995) Development and

analysis of retroviral vectors expressing human factor VIII as a potential gene therapy for

hemophilia A Hum Gene Ther 6: 1363 – 1377.

16 Dwarki, V J., et al (1995) Gene therapy for hemophilia A: production of therapeutic

levels of human factor VIII in vivo in mice Proc Natl Acad Sci USA 92: 1023 – 1027.

17 Krall, W J., et al (1996) Increased levels of spliced RNA account for augmented

expression from the MFG retroviral vector in hematopoietic cells Gene Ther 3:

37 – 48.

18 Swaroop, M., Moussalli, M., Pipe, S W., and Kaufman, R J (1997) Mutagenesis of a

potential immunoglobulin-binding protein-binding site enhances secretion of

coagu-lation factor VIII J Biol Chem 272: 24121 – 24124.

19 Kootstra, N A., Matsumura, R., and Verma, I M (2003) Efficient production of human

factor VIII in hemophilic mice using lentiviral vectors Mol Ther 7: 623 – 631.

20 Qian, J., Collins, M., Sharpe, A H., and Hoyer, L W (2000) Prevention and treatment

of factor VIII inhibitors in murine hemophilia A Blood 95: 1324 – 1329.

21 Pittman, D D., et al (1993) Biochemical, immunological, and in vivo functional

characterization of B-domain-deleted factor VIII Blood 81: 2925 – 2935.

22 Kaufman, R J., Wasley, L C., and Dorner, A J (1988) Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells J Biol Chem 263: 6352 – 6362.

23 Tonn, T., Herder, C., Becker, S., Seifried, E., and Grez, M (2002) Generation and characterization of human hematopoietic cell lines expressing factor VIII J Hematother Stem Cell Res 11: 695 – 704.

24 Tiede, A., et al (2003) Recombinant factor VIII expression in hematopoietic cells following lentiviral transduction Gene Ther 10: 1917 – 1925.

25 Kaneko, S., Onodera, M., Fujiki, Y., Nagasawa, T., and Nakauchi, H (2001) Simplified retroviral vector GCsap with murine stem cell virus long terminal repeat allows high and continued expression of enhanced green fluorescent protein by human hematopoietic progenitors engrafted in nonobese diabetic/severe combined immuno-deficient mice Hum Gene Ther 12: 35 – 44.

26 Chuah, M K L., et al (2000) Long-term persistence of human bone marrow stromal cells transduced with factor VIII-retroviral vectors and transient production of therapeutic levels of human factor VIII in nonmyeloablated immunodeficient mice Hum Gene Ther 11: 729 – 738.

27 Van Damme, A., et al (2003) Bone marrow mesenchymal cells for haemophilia A gene therapy using retroviral vectors with modified long-terminal repeats Haemophilia 9:

94 – 103.

28 Kalberer, C P., et al (2000) Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human beta-globin in engrafted mice Proc Natl Acad Sci USA 97:

5411 – 5415.

29 Israel, D I., and Kaufman, R J (1990) Retroviral-mediated transfer and amplification of

a functional human factor VIII gene Blood 75: 1074 – 1080.

30 Miao, H Z., et al (2004) Bioengineering of coagulation factor VIII for improved secretion Blood 103: 3412 – 3419.

31 Bunting, K D., and Hawley, R G (2003) Integrative molecular and developmental biology of adult stem cells Biol Cell 95: 563 – 651.

32 Koc, O N., et al (1999) Bone marrow-derived mesenchymal stem cells remain host-derived despite successful hematopoietic engraftment after allogeneic transplantation

in patients with lysosomal and peroxisomal storage diseases Exp Hematol 27:

1675 – 1681.

33 Sun, S., et al (2003) Isolation of mouse marrow mesenchymal progenitors by a novel and reliable method Stem Cells 21: 527 – 535.

34 Ally, B A., et al (1995) Prevention of autoimmune disease by retroviral-mediated gene therapy J Immunol 155: 5404 – 5408.

35 Kang, E., et al (2001) In vivo persistence of retrovirally transduced murine long-term repopulating cells is not limited by expression of foreign gene products in the fully or minimally myeloablated setting Hum Gene Ther 12: 1663 – 1672.

36 Puig, T., et al (2002) Myeloablation enhances engraftment of transduced murine hematopoietic cells, but does not influence long-term expression of the transgene Gene Ther 9: 1472 – 1479.

37 Andersson, G., et al (2003) Engraftment of retroviral EGFP-transduced bone marrow

in mice prevents rejection of EGFP-transgenic skin grafts Mol Ther 8: 385 – 391.

38 Heim, D A., et al (2000) Introduction of a xenogeneic gene via hematopoietic stem cells leads to specific tolerance in a rhesus monkey model Mol Ther 1: 533 – 544.

39 Kung, S K., et al (2003) Induction of transgene-specific immunological tolerance in myeloablated nonhuman primates using lentivirally transduced CD34 + progenitor cells Mol Ther 8: 981 – 991.

40 Mardiney, M.,3 rd , and Malech, H L (1996) Enhanced engraftment of hematopoietic progenitor cells in mice treated with granulocyte colony-stimulating factor before low-dose irradiation: implications for gene therapy Blood 87: 4049 – 4056.

41 Hacein-Bey-Abina, S., et al (2003) LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1 Science 302: 415 – 419.

42 McCormack, M P., and Rabbitts, T H (2004) Activation of the T-cell oncogene LMO2 after gene therapy for X-linked severe combined immunodeficiency N Engl J Med 350: 913 – 922.

43 Hawley, R G., Covarrubias, L., Hawley, T., and Mintz, B (1987) Handicapped retroviral vectors efficiently transduce foreign genes into hematopoietic stem cells Proc Natl Acad Sci USA 84: 2406 – 2410.

44 Ramezani, A., Hawley, T S., and Hawley, R G (2003) Performance- and safety-enhanced lentiviral vectors containing the human interferon-h scaffold attachment region and the chicken h-globin insulator Blood 101: 4717 – 4724.

45 Hawley, R G., Lieu, F H., Fong, A Z., Goldman, S J., Leonard, J P., and Hawley, T S (1996) Retroviral vectors for production of interleukin-12 in the bone marrow to induce a graft-versus-leukemia effect Ann N Y Acad Sci 795: 341 – 345.

46 Lindemann, D., Patriquin, E., Feng, S., and Mulligan, R C (1997) Versatile retrovirus vector systems for regulated gene expression in vitro and in vivo Mol Med 3: 466 – 476.

47 Hawley, T S., Sabourin, L A., and Hawley, R G (1989) Comparative analysis of retroviral vector expression in mouse embryonal carcinoma cells Plasmid 22: 120 – 131.

48 Karasuyama, H., and Melchers, F (1988) Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors Eur J Immunol 18: 97 – 104.

49 Harris, J F., Hawley, R G., Hawley, T S., and Crawford-Sharpe, G C (1992) Increased frequency of both total and specific monoclonal antibody producing hybridomas using