ARTICLE doi:10.1016/j.ymthe.2004.03.005

Efficient Lentiviral Vector-Mediated Control of HIV-1 Replication in CD4 Lymphocytes from Diverse HIV+ Infected Patients Grouped According to CD4 Count and Viral Load

Laurent M. Humeau,1,* Gwendolyn K. Binder,1,* Xiaobin Lu,1 Vladimir Slepushkin,1 Randall Merling,1 Patricia Echeagaray,1 Mario Pereira,1 Tatiana Slepushkina,1 Scott Barnett,2 Lesia K. Dropulic,3 Richard Carroll,4 Bruce L. Levine,4 Carl H. June,4 and Boro Dropulic1,5,y

1 VIRxSYS Corporation, Gaithersburg, MD 20877, USA 2 The Gary Lambert Research Center, Johns Hopkins University, Baltimore, MD 21287, USA 3 Department of Medicine, The Johns Hopkins University Division of Infectious Diseases, Baltimore, MD 21205, USA 4 Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104, USA 5 Sydney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA

*These authors contributed equally to this work.

yTo whom correspondence and reprint requests should be addressed at VIRxSYS Corporation, 200 Perry Parkway, Suite 1A, Gaithersburg, MD 20877. Fax: (301) 987-0489. E-mail: [email protected] or [email protected].

Available online 7 May 2004

We present preclinical studies that demonstrate in vitro the feasibility and efficacy of lentivirus- based vector antisense gene therapy for control of HIV replication in primary T lymphocytes isolated from HIV-infected patients discordant for clinical status. VRX496 is a VSV-G-pseudotyped HIV-based vector that encodes an antisense payload against the HIV envelope gene. The antisense payload is under the control of the native LTR promoter, which is highly transactivated by tat upon HIV infection in the cell. Transfer of autologous CD4+ T lymphocytes genetically modified with VRX496 (VRX496T) into HIV-infected patients is intended to provide a reservoir of cells capable of controlling HIV, potentially delaying AIDS onset. To determine the patient population likely to respond to VRX496 for optimal efficacy, we examined the ability of our research vector, VRX494, to modify and suppress HIV in vitro in lymphocytes isolated from 20 study subjects discordant for CD4 count and viral load. VRX494 is analogous to the clinical vector VRX496, except that it contains GFP as a marker gene instead of the 186-tag marker in the clinical vector. To transfer VRX494 to target cells we developed a novel scalable two-step transduction procedure that has been translated to the clinic in an ongoing clinical trial. This procedure achieved unprecedented transduction efficiencies of 94 F 5% in HIV+ study subject cells. In addition the vector inhibited HIV replication z93% in culture regardless of the viral load or CD4 count of the subject or tropism of the virus strain with which they were infected. These findings demonstrate that VRX496T therapy is expected to be beneficial to patients that differ in their status in term of CD4 count and viral load. The methods described represent significant technical advances facilitating execution of lentivirus vector-mediated gene therapy for treatment of HIV and are currently being employed in the first trial evaluating lentivirus vector safety in humans.

Key Words: HIV-1, gene therapy, viral RNA, lentivirus, genetic vectors, clinical trials, CD4-positive T lymphocytes, acquired immunodeficiency syndrome

ically consists of a triple ‘‘cocktail’’ of a nucleoside reverse INTRODUCTION transcriptase inhibitor, a nonnucleoside reverse transcrip- The current standard of treatment for HIV/AIDS is highly tase inhibitor, and a protease inhibitor. Although HAART active antiretroviral therapy (HAART). This therapy typ- has been successful in reducing viral loads and restoring

902 MOLECULAR THERAPY Vol. 9, No. 6, June 2004 Copyright B The American Society of Gene Therapy 1525-0016/03 $30.00 doi:10.1016/j.ymthe.2004.03.005 ARTICLE

immune function, it does not represent a cure and it is genetic vectors, including HIV-1-based lentiviral vectors, now estimated that an average patient would require and accompanying genetic antiviral payloads have been anywhere from 51 to 73 years to purge a latent memory proposed to combat HIV-1, including antisense RNA, T lymphocyte reservoir [1,2]. transdominant proteins, ribozymes, RNA decoys, single- There are concerns regarding the adverse effects asso- chain antibodies, and RNAi [15–18].AntisenseRNA ciated with HAART usage, and for many HIV+ patients, targeted to wild-type (wt) HIV RNA offers a significant treatment does not provide a feasible long-term solution advantage over several other genetic antiviral [3–5]. Significant levels of adverse effects are associated approaches since it is not a protein and thus not immu- with each of the HAART drug groups, which range be- nogenic, and unlike with ribozymes or RNAi, the size of tween 1 and 30% of patients for each group [6]. Upon the payload prevents development of escape mutant considering the percentage of adverse events when taking viruses [19]. Although several of these approaches have three drugs together, it then becomes evident that man- been tested in vitro during virus challenge, no study has agement of HIV with HAART is difficult for a majority of ever demonstrated efficacy in primary HIV+ patient patients. Combined with complex and cumbersome dos- lymphocytes using a clinically relevant vector and gene ing regimens, HAART can have a negative impact on transfer protocol. patient–subject adherence to therapy [6,7]. VRX496 is the first HIV-1-based lentiviral vector to be The need for novel HIV therapeutics is evident. Poor evaluated for safety in humans. VRX496 is a fully gutted adherence to HAART has led to an increased rate of HIV vector derived from HIVNL4-3 [20] that does not encode resistance, resulting in viral strains that have reduced any viral proteins. VRX496 was designed in parallel with sensitivity to the drugs [8,9]. As many as 18.5% of newly the helper plasmid with several safety features to limit transmitted HIV infections in the United States are resis- the likelihood of a possible recombination event in vivo tant to combination antiretroviral drug therapy and that could lead to a replication-competent lentivirus, or transmitted resistance has been increasing significantly RCL [21]. VRX494 is the research analogue to VRX496 since 1995 [10,11]. These patients have no treatment and is identical except that it additionally encodes GFP alternatives and have very poor prognoses, which has as a marker gene downstream of the antisense payload. prompted the need for new anti-HIV drugs active against VRX494 and VRX496 encode a 937-nucleotide antisense resistant strains to become available rapidly. The most sequence targeted to the HIV-1 envelope (env) gene recent new antiretroviral drug active against such resis- (Fig. 1). tant strains, a fusion inhibitor, works in few patients with VRX496 is intended for ex vivo transduction of autol- a low efficacy, high cost, and high risk of side effects [12]. ogous T lymphocytes from HIV-infected patients. To The fact that such a drug is considered an acceptable ensure therapeutic efficacy in the clinic, several points alternative for HIV treatment underscores the urgent of progress had first to be made. The first of these was to need for novel approaches for treatment. achieve efficient and modulatory gene transfer into HIV+ Gene therapy for HIV-1 infection, also referred to as lymphocytes and demonstrate that VRX496 was effica- intracellular immunization, was first proposed at least as cious against autologous wt-HIV replication (information early as 1989 [13] and has been suggested as an alterna- regarding the modulatory nature of transduction using tive to HAART therapy [14,15]. A number of different VRX496 is presented in Ref. [21]). Second, to facilitate

FIG. 1. Schematic representation of HIV and the corresponding regions derived for vector construc- tion. The NL4-3 strain of HIV is shown, along with the clinical vector, VRX496, and a GFP-expressing re- search vector, VRX494. The two vectors are identical, except that VRX496 contains a molecular tag region in place of the GFP gene. The 937-base antisense payload is under the control of the LTR and is splice- dependent. A central polypurine tract (cppt) se- quence is included to facilitate nuclear entry. The rev-response element (RRE) is also included to facilitate RNA export to the cytoplasm. The pack- aging sequence is retained from gag (psi).

MOLECULAR THERAPY Vol. 9, No. 6, June 2004 903 Copyright B The American Society of Gene Therapy ARTICLE doi:10.1016/j.ymthe.2004.03.005

selective outgrowth of HIV-resistant cells, a survival ad- was a result of a slower response of the infected cells to vantage of transduced over untransduced cells during bead activation and that transduction could be improved HIV infection in vitro must be established. Finally, a by adding vector later in culture after more cells had been scalable and clinically translatable transduction proce- more fully activated and synchronized. To test this, we dure must be developed to apply this therapy in patients. added half of the vector on the first day of the culture and After transduction, modified lymphocytes (VRX496T) the other half on the second day of culture, for a total will be subsequently reintroduced into the patient intra- amount of vector not exceeding that which was used in venously. Modified lymphocytes may decrease viral loads normal donor cells (20 TU per cell). After this protocol in vivo, particularly if they resist productive HIV infection adjustment, transduction efficiencies consistently in- and selectively expand and repopulate long term in the creased, usually to z90%, levels similar to those of body, which may in turn alleviate the symptoms of normal donor cells (Table 1, Fig. 2). An examination of disease caused by HIV/AIDS [22–24]. transduction efficiencies according to patient group The first clinical trial using a lentivirus-based vector reveals that permissiveness to vector transduction is for gene transfer began in January 2003, with the goal of independent of viral load and CD4 count. All 17 patients determining the safety and tolerability of VRX496 in a tested using the optimized two-dose protocol were trans- very narrow range of HIV-infected patients–subjects [25]. duced at high efficiency. We present in this report preclinical studies demonstrat- It is important here to acknowledge that stimulation ing a scalable and clinically relevant gene transfer pro- of HIV-infected cells with anti CD3/CD28 antibodies can cedure that uses the GFP-expressing analogue to activate CXCR4-tropic, but not CCR5-tropic HIV replica- VRX496, VRX494, to transduce primary CD4+ T lympho- tion. Therefore, as a safety precaution in our ongoing cytes efficiently and inhibit autologous HIV replication clinical trial, cells are cultured in the presence of antire- in vitro in a diverse HIV-infected study subject population troviral drugs. As part of the release testing, expanded grouped into three groups according to CD4 count and modified cells are tested postexpansion to ensure that viral load. there has been no increase in proviral copy number.

Antisense-Mediated Protection from HIV Challenge in RESULTS Normal CD4+ T Lymphocytes Method for High-Efficiency VRX494-Mediated To evaluate the role of antisense in controlling HIV Transduction of HIV+ CD4+ T Lymphocytes replication, we examined the relative contributions of For an efficient, scalable, and clinically translatable pro- the vector backbone and antisense payload to the anti- cedure, healthy donor peripheral blood mononuclear HIV effect. We transduced the T cell line SupT1 at 20 cells (PBMCs) are collected and purified for CD4+ T TU per cell with VRX494 or an empty control vector lymphocytes by MACS positive selection. Cells are mixed identical to VRX494 without the 937-base antisense with anti-CD3/28 antibody-coated beads at a ratio of 3 sequence or transduced cells with a buffer control. beads/cell and then are typically added to a Retronectin- We then challenged transduced cells with HIV at a coated plate that has been preincubated for 30 min with multiplicity of infection (m.o.i.) of 0.1. We measured vector at a concentration of 20 transducing units (TU) per virus replication by ELISA for p24 protein in the cell. Cultures are carried for 3 days in medium containing culture supernatant (Fig. 3A), and detected peak p24 human serum, antibiotics, and interleukin (IL)-2 (see levels in mock-transduced cells about 7 days postchal- Methods). At day 3, cells are dispersed, washed of vector, lenge. We observed a marginally lower p24 peak 5 days and replated at a concentration of f0.3 to 0.5 106 cells/ later in the cultures transduced with the empty control ml. Seven days after culture initiation (4 days after the vector. Despite the delayed p24 peak, we observed no wash), cells are dispersed and separated from the beads protection as the empty vector cultures were obliterated and then expanded as necessary. This protocol has by the end of the experiment like the mock-transduced achieved greater than 95% transduction efficiency in cultures. In contrast, VRX494 protected SupT1 cells normal CD4+ T lymphocytes [21]. Additional informa- throughout the duration of the culture, despite the tion regarding the effects of CD3/28 expansion on lym- high m.o.i. challenge. This experiment demonstrates phocytes, for example examination of the Vh repertoire, the specific role of the antisense payload in conferring may be found in Levine et al. [26]. anti-HIV resistance. Since HIV-infected cells express lower levels of CD28 To determine the ability of VRX494 to inhibit several and respond less well to immobilized (i)CD3 and mitogen common strains of HIV, we transduced healthy donor [27–29], we expected that transduction efficiencies in primary CD4+ T lymphocytes at 20 TU per cell and patient cells would be lower than those in healthy cells. subsequently infected them at high m.o.i. of 0.01 with We indeed found this to be the case, and our first three CXCR4 (X4), CCR5 (R5) or an X4/R5 combination strain patients exhibited low transduction efficiencies ranging of HIV. In addition to the stimulatory beads added at from 25 to 67%. We hypothesized that this low efficiency culture initiation as described under Methods, we intro-

904 MOLECULAR THERAPY Vol. 9, No. 6, June 2004 Copyright B The American Society of Gene Therapy doi:10.1016/j.ymthe.2004.03.005 ARTICLE

TABLE 1: HIV+ study subject groups and associated vector transduction efficiencies compared to parallel HIV-negative donor controlsa Group Patient Viral load CD4 count % Td Pat. % Td ND Cntlc (copies/ml plasma) (cells/mm3 blood) dayb 0 day 0 (1) Viral loadd J21 124,949 321 87 99 >50,000; J25 222,612 403 96 99 CD4e 200–600 J28 95,591 288 96 93 J33 163,808 232 93 99 (2) Viral load J08 19,170 499 80 98 500–50,000; J19 22,847 299 93 98 CD4 200–600 J20 35,292 578 92 99 J23 1,230 347 97 99 J24 1,400 300 98 99 J29 9,279 305 90 93 J31 15,732 359 96 98 U01 18,500 415 98 98 (3) Viral load J05 4,487 664 92 98 500–50,000; J22 24,815 758 99 99 CD4 >600 J30 12,578 765 88 93 J32 36,763 995 98 98 J34 23,891 1214 99 99 a The first three patients tested with the original transduction protocol (one vector addition) are not listed. b Percentage of transduced patient (Pat.) cells on day 0. c Percentage of transduced normal donor cells (ND) on day 0. d Viral load expressed as RNA copies per milliliter of plasma. e CD4 count expressed as cells per cubic millimeter of blood.

duced additional beads into cultures at days 4 and 16 As a result, transduced CD4+ T lymphocytes exhibited postchallenge (at 1 bead per cell) to stimulate expansion a selective advantage for growth in culture after challenge of the culture continually for long-term analysis. We with wt-HIV. We have established that increased copy measured HIV virion production in culture supernatants numbers correlate directly with enhanced GFP mean by p24 ELISA for 34 days postchallenge. VRX494 fluorescence intensity (MFI) (unpublished data). At day inhibited virion production in all cultures. The degree 36, we observed a decrease in the number of nontrans- of suppression varied between individual strains, but was duced cells and a virtual disappearance of cells trans- independent of virus tropism (Fig. 3B). duced at lower copy numbers, or MFI, with VRX494, Downregulation of CD4 surface marker expression is while those transduced at greater copy numbers, or at a indicative of productive HIV infection, resulting from greater MFI, were enriched (Fig. 3D). Therefore, the expression of HIV nef, gp120, or vpu proteins [30]. selective survival of cells in vitro with multiple copies Therefore, CD4 expression can serve as a surrogate mark- capable of surviving an intense challenge illustrates the er for vector-mediated control of HIV replication. To selective advantage conferred by the antisense-contain- determine whether VRX494 could provide selective resis- ing vector. tance to HIV-mediated downregulation of CD4 surface To investigate further vector-mediated selective ad- marker expression, we minimally transduced CD4+ T vantage, we transduced healthy donor CD4+ T lympho- lymphocytes isolated from healthy donors with VRX494 cytes with our clinical grade vector, VRX496, at 5, 10, or to yield f50% transduced cells in culture. We then 20% of total culture volume and challenged cultures with infected the cultures at an m.o.i. of 0.05 with the R5 HIVNL4-3 at a more clinically relevant m.o.i. of 0.001. US1 strain of HIV. This mixed transduced and nontrans- These percentages correlate with approximately 0.9, 1.9., duced culture allows for a dynamic infection of the and 3.8 TU/cell. Typically, the potency of clinical grade unprotected cells while rigorously challenging the pro- vector is established in primary T cells using 5, 10, and tected (transduced) cells for the purposes of analysis. 20% culture transduction. Potency is established this Nontransduced control cells exhibited downregulation way, instead of a ‘‘per TU’’ assessment, because in con- of surface CD4 when analyzed at day 36 postinfection, trast to centrifuged vector, the clinical purification pro- while GFP-positive cells in the transduced cultures cess reduces the potency of the vector to approximately exhibited little to no downregulation. GFP-negative cells 30% of the original potency, so that achieving target (nontransduced) in the transduced culture also had re- copy numbers of 0.5–5 copies per cell requires 20% of duced CD4 surface expression (Fig. 3C). the culture volume. Therefore, for all experiments using

MOLECULAR THERAPY Vol. 9, No. 6, June 2004 905 Copyright B The American Society of Gene Therapy ARTICLE doi:10.1016/j.ymthe.2004.03.005

FIG. 2. Vector transduction in study subject cells. Cells were mock transduced or transduced in parallel with healthy donor controls. The percentage of events in each quadrant is shown, and the patient viral load and CD4 count are shown to the right of the patient panel. Cultures were analyzed on day 7 after bead removal (day 14 of culture).

clinical vector, cells will be transduced at a percentage of challenged at an m.o.i. of 0.001, and virus replication was the culture volume instead of a specific TU dose per cell. evaluated by ELISA for p24 production. We found that Initially, all cultures expanded similarly as the rate of transduced cultures were able to reduce virus replication HIV-induced cell death did not surpass the rate of cell over 3 logs compared to untransduced controls. Togeth- growth following activation. However, at peak HIV rep- er, these data show that VRX496 inhibits HIV replication lication as measured by p24 production at day 10, cell up to almost 99.99%, or 4 logs, in several strains of HIV in death in nontransduced cultures exceeded or equaled cell CD4+ T lymphocytes and that this inhibition is concur- growth thus reducing cumulative culture expansion. We rent with decreased CD4 downregulation and a selective found a vector dose-dependent selective growth advan- growth advantage. tage in transduced cultures after in vitro challenge, which was accordingly consistent with increases in vector copy Inhibition of Endogenous HIV Replication in Patient number (Fig. 3E). Lymphocytes Finally, we investigate the ability of our clinical-grade To evaluate VRX494 in lymphocytes isolated from HIV+ vector, VRX496, to protect primary CD4+ T lymphocytes individuals, we selected patients that fell into one of from HIV infection (Fig. 3F). Cultures were transduced at three groups: high viral load and low CD4 count, low 20% culture volume with VRX496, or vector storage viral load and high CD4 count, and low viral load and buffer alone. Seven days posttransduction, cultures were low CD4 count. No patients with a high viral load and

FIG. 3. Vector-mediated inhibition of HIV. (A) Cells from the T cell line, SupT1, were transduced with VRX494 (squares) or VRX494 without antisense (closed circles), or mock transduced (open circles). Three days later cells were challenged with HIVNL4-3 at an m.o.i. of 0.1. Cell counts were normalized at each passage when replated. (B) CD4+ T lymphocytes were transduced (closed squares) or mock transduced (open squares) and subsequently infected with CXCR4 (X4), CCR5 (R5), or X4R5 strains of HIV. Specific virus strains are listed above each graph. Standard deviation is represented by error bars (n = 3). (C, D) Selective advantage of transduced cells after HIV infection. (C) Inhibition of CD4 downregulation in CD4+ T lymphocytes transduced with VRX494 after HIV challenge. FACS analysis was performed at day 36 post-HIV challenge. Shown are HIV-negative and HIV strain US1-challenged mock and transduced cultures as indicated. In the upper right corner is shown the percentage of cells in each quadrant. (D) Mean fluorescence intensity in partially transduced (50%) CD4-gated cultures 36 days following HIV infection (shaded line) or mock infection (open line). (E) Fold cell expansion in mock (open symbols) and 5% v/v, 10% v/v, and 20% v/v clinical vector VRX496-transduced cultures after challenge with HIVNL4-3. All cultures were transduced at 90% or greater, but the resulting copy numbers increased with increased vector addition. Corresponding copy numbers per cell are represented in the legend next to each percentage. (F) VRX496-mediated inhibition of HIV after challenge in primary CD4+ T lymphocytes. Transduced (closed symbols) or mock-transduced (open symbols) cultures were challenged with HIVNL4-3 at an m.o.i. of 0.001. HIV replication was measured by p24 ELISA and results were normalized to cell count.

906 MOLECULAR THERAPY Vol. 9, No. 6, June 2004 Copyright B The American Society of Gene Therapy doi:10.1016/j.ymthe.2004.03.005 ARTICLE

high CD4 count could be identified during the period expression of CD4 surface marker, and cell expansion. of the study. We transduced cells isolated from these We observed a steady and continuous decrease in HIV patients, at 20 TU per cell as described in the first virion production as measured by p24 ELISA through- results section. Cultures were monitored for HIV repli- out the culture period in transduced patient cells com- cation by intracellular p24 and secretion into culture, pared to increasing virion production in untransduced

MOLECULAR THERAPY Vol. 9, No. 6, June 2004 907 Copyright B The American Society of Gene Therapy ARTICLE doi:10.1016/j.ymthe.2004.03.005

cells (Fig. 4A, left). This correlated with reduced per- lymphocyte markers (CD45-RO and CD45-RA, respective- centages of HIV+ lymphocytes in transduced cultures, ly) at each passage. We measured the activation marker as detected by intracellular p24 staining, indicating that CD28 and the suppression marker CTLA-4 at culture vector inhibition of virus replication also prevents initiation and termination. We did not observe differ- spread of HIV in culture (Fig. 4A, right). A summary ences in cell surface marker expression between trans- of p24 production and percentage inhibition over con- duced and mock cultures with any of the markers trols and percentages of infected cells in all patients examined (data not shown). examined is presented in Table 2. Values in this table Finally, to determine whether VRX494 could inhibit were taken late in culture (day 10 or later), at the point replication of different strains of HIV in study subjects when the highest level of HIV replication was detected we examined the tropism of the virus in each of the in mock-transduced cultures, since this would be the patients studied using the GHOST cell assay described in point at which vector inhibition of HIV could be most Morner et al. [31]. We found that VRX494 is capable of rigorously determined. Inhibition ranged from 93 to controlling replication of both X4 and R5 primary 100% between patients, except for patients J23 and strains of HIV among the patients tested in this study. J34, in which no virus replication could be detected Therefore this supports the rationale for including early even in mock-transduced cultures. Inhibition of p24 in and late stage HIV-infected patients in VRX496T therapy culture supernatants correlated directly with lower per- (Table 3). centages of cells infected with HIV in transduced com- pared to mock cultures. The consistent and high level of inhibition of virion production observed in trans- DISCUSSION duced patient cultures (on average 98% for each group) Transfer of autologous CD4+ T lymphocytes modified was independent of the patient’s viral load or CD4 with an anti-HIV genetic payload is intended to result count. in reduced viral loads in vivo thus temporarily or Productive HIV replication results in cellular dysfunc- permanently removing the need to suppress virus rep- tion in part through cell death by apoptosis and down lication with antiretroviral drugs. Vector-mediated in- regulation of the CD4 receptor [30]. HIV study subject hibition of HIV replication may prevent the death of cultures transduced with VRX494 exhibited decreased HIV-infected cells and/or reduce the spread of the virus cell death, as measured indirectly by a significant increase by inhibiting virus release and protecting uninfected in cumulative cell expansion that did not exceed that of cells from de novo infection, thus potentially postpon- uninfected mock or transduced cultures. Over half of the ing the onset of AIDS. Resistance to HIV replication transduced cultures expanded more than the mock-trans- may also lead to a selective outgrowth of transduced duced controls (Fig. 4B, Table 3). The endogenous level of cells, which could improve patient health by providing HIV infection was not high enough and long enough to them with a reservoir of CD4+ T lymphocytes protected demonstrate the vector-mediated selective advantage in from HIV that could then contribute to the anti-HIV all study subject lymphocyte cultures. At the end of the immune response. culture period, the cells were dividing very slowly; to Precedent for such selective expansion of gene-modi- prolong the culture for examination of a selective advan- fied cells is provided by the fact that immune reconsti- tage over the longer term, the cells would have had to tution was promoted in a child with adenosine have been stimulated again by beads. We did not do this deaminase (ADA) deficiency after removal from enzyme since it would not have provided information over that replacement therapy resulting from toxicity [32]. Prior to presented in Figs. 3C–3E in terms of anti-HIV efficacy in removal from replacement therapy, infused transduced patient cells, since the dynamics of selection in vivo are lymphocytes containing a transgene for adenosine deam- expected to be far different than that in vitro. In study inase persisted in vivo at 1–3%. However, after removal subject cultures in which HIV-mediated downregulation from therapy, transduced cells exhibited a remarkable of CD4 occurred, transduced cultures displayed decreased selective growth advantage that resulted in a stable T downregulation compared to mock cultures (Fig. 4B, lymphocyte immune repopulation approaching 100%, Table 3). CD4 expression was not downregulated in every and the patient exhibited persistent and normal levels patient culture. These data further demonstrate VRX494- of ADA enzyme. mediated inhibition of HIV replication in a broad range After validation of the anti-HIV efficacy of VRX494 of patients. in challenge experiments using various strains of HIV In addition to CD4, we monitored other common in T cells isolated from normal subjects, we demon- CD4+ T lymphocyte surface markers as a general mea- strated vector-mediated control of HIV in primary surement for whether treatment of cells with vector was human CD4+ T lymphocytes derived from a range of affecting T lymphocyte status. We examined the cell study subjects. Using several parameters to measure surface marker expression for the IL-2 receptor subunits HIV replication, we determined that VRX494 is capable a and h (CD25 and CD122) and memory and naı¨ve T of efficient transduction (94 F 5%) and subsequent

908 MOLECULAR THERAPY Vol. 9, No. 6, June 2004 Copyright B The American Society of Gene Therapy doi:10.1016/j.ymthe.2004.03.005 ARTICLE

FIG. 4. Inhibition of HIV replication in study subject cells. (A) Inhibition of p24 production. Mock (open symbols) and transduced (closed symbols) cultures from patients U01, J25, and J28 were examined for extracellular p24, a surrogate marker for HIV virion production (left). Each mock and transduced culture was carried in triplicate, and each triplicate well was measured by ELISA in triplicate. Intracellular p24, which quantitates the number of HIV-infected cells in a culture and reflects HIV spread in culture, was also performed (right). Error bars represent SEM determined for each triplicate culture using the average ELISA values for each triplicate (left). (B) Expansion of and CD4 expression on transduced patient CD4+ T lymphocyte cultures. (Left) HIV+ study subject T cells transduced with VRX494 have a selective advantage over mock-transduced cells, which does not exceed expansion of normal donor cells. Transduced cultures (closed) and mock controls (open) for three representative study subjects, J33, J31, and J22 (squares), and a healthy donor control (circles) are shown. Day 7 represents culture initiation, and day 0 represents the day that the beads were removed from cells. Patient viral loads and CD4 counts are indicated on the left. (Right) HIV- mediated CD4 downregulation is inhibited by VRX494. Study subjects (squares) are shown with parallel HIV-negative controls (circles). Mock (open symbols) and transduced (closed symbols) cultures were carried in triplicate. Cells were collected at each culture passage and samples from triplicate cultures were pooled and imaged by flow cytometry after staining for CD4 antigen, so no error bars are shown.

MOLECULAR THERAPY Vol. 9, No. 6, June 2004 909 Copyright B The American Society of Gene Therapy ARTICLE doi:10.1016/j.ymthe.2004.03.005

TABLE 2: Inhibition of HIV replication and spread in VRX494-transduced cultures in comparison to mock-transduced study subject cultures Group Patient p24 (pg per million cells) % Inhibition % p24-positive cells Avg % Inhibition ID per group Mock Transduced Mock Transduced (1) Viral load J21 53,883.7 15.3 99.97 2.20 0.02 Avg >50,000; J25 4,488.5 2.7 99.94 2.86 0.05 98.265 CD4 200–600 J28 30,464.4 0.7 100 4.60 0.05 SD J33 7,580,400 76,545.8 93.15 43.52 15.02 3.410088 (2) Viral load J08 213.7 5.3 97.52 11.71 10.78 Avg 500–50,000; J19 42,854.9 763.2 98.22 16.07 7.89 98.06857 CD4 200–600 J20 38,289.4 667.4 98.26 24.22 2.43 SD J23 0.0 0.0 NA 0.02 0.04 1.793511 J24 18,399.9 130.0 99.29 6.14 0.15 J29 2.8 0.1 94.43 0.02 0.06 J31 70,459.0 876.0 98.76 10.46 0.92 U01 7,087.6 0.1 100 2.99 0.04 (3) Viral load J05 81.6 1.7 97.94 0.15 0.07 Avg 500–50,000; J22 27,117.2 47.4 99.83 8.24 0.05 97.8425 CD4 >600 J30 48,719.6 3,105.2 93.63 8.25 2.27 SD J32 964.8 0.3 99.97 0.79 0.01 2.956973 J34 0 0 NA 0.15 0.11 inhibition of HIV replication (z93%) regardless of virus the antisense sequence and the infecting virus envelope. tropism in all patients. This is supported by Fig. 3A in which R5, X4, and R5/X4 In challenge experiments as well as in primary CD4+ T strains are similarly inhibited (left). Tropism is primarily lymphocytes isolated from HIV+ patients, we observed determined by the V3 loop region of the envelope [33], varying levels of virus inhibition as measured by virion which is targeted by the vector expressing antisense, production in culture supernatants. This variation is therefore the various strains tested here are likely to have likely a reflection of the virulence of the infecting HIV sequence differences within the antisense target region. strain and not a result of the level of homology between Yet the efficacy of the antisense was similar between

TABLE 3: Expansion, CD4 cell surface marker downregulation, and HIV tropism in study subject CD4+ T lymphocyte cultures Group Patient Viral load CD4 count Relative expansion P valueb % CD4 % CD4 Tropism ID (copies/ml plasma) (cells/mm3 blood) (Td/M)a mock transduced (1) Viral load J21 124,949 321 1.6 0.017 84 88 R5 >50,000; J25 222,612 403 1 0.857 100 100 R5 CD4 200–600 J28 95,591 288 0.9 0.85 88 99 R5 J33 163,808 232 7.6 < 0.0009 84 92 X4 (2) Viral load J08 19,170 499 1.3 0.199 72 78 NDc 500–50,000; J19 22,847 299 2.6 < 0.0009 91 97 R5 CD4 200–600 J20 35,292 578 2.6 < 0.0009 42 58 R5 J23 1,230 347 0.9 0.85 96 100 R5 J24 1,400 300 1.6 0.017 98 100 R5 J29 9,279 305 1.4 0.098 100 100 ND J31 15,732 359 3.7 < 0.0009 85 100 X4 U01 18,500 415 1.2 0.359 98 100 ND (3) Viral load J05 4,487 664 0.9 0.85 98 99 ND 500–50,000; J22 24,815 758 1.5 0.043 90 99 R5 CD4 >600 J30 12,578 765 3.4 < 0.0009 83 96 X4 J32 36,763 995 3.7 < 0.0009 100 100 ND J34 23,891 1214 1.3 0.199 92 92 ND a Fold-expansion rate was determined at the final day of culture (day 17) and then the values from transduced cultures were divided by mock values to get the ratio presented. b A t test was performed to determine whether the fold-expansion rate in transduced cultures was significantly greater than that of mock cultures. c ND, not determined by GHOST cell assay for virus tropism as described in [31].

910 MOLECULAR THERAPY Vol. 9, No. 6, June 2004 Copyright B The American Society of Gene Therapy doi:10.1016/j.ymthe.2004.03.005 ARTICLE

study subjects. We previously conducted an in-depth against the envelope gene, which would reduce the level study examining the ability of HIV to develop escape of envelope on mobilizing vector particles and hence mutants to evade antisense therapy and found that HIV their infectivity. Indeed, we have observed vector being either deletes significant portions of the envelope region produced by cells infected with HIV, but those vector or incorporates high levels of point mutations in the particles are deficient in secondary transduction. These antisense target region that leave the virus severely data may be reviewed in Lu et al. [21]. debilitated and unable to replicate in the presence of These studies demonstrate for the first time that gene antisense [19]. therapy for HIV using a clinically applicable vector and HIV-resistant cells exhibited a selective advantage gene transfer protocol is effective in controlling virus in a both after challenge and in primary study subject lym- range of primary lymphocytes isolated from HIV+ phocytes. A selective advantage in culture is more clear- patients. The studies indicate that an HIV-1 vector-medi- ly observed after challenge since it introduces higher ated genetic therapeutic approach for treatment of HIV is levels of virus into the cells than what is usually ob- feasible, effective, and applicable across a broad patient served during natural infection, in which as few as 1 in population discordant in disease status and virus tropism. 2800 cells may harbor provirus at one time (an m.o.i. of This represents an encouraging new therapeutic modality 0.00034) [34]. Not all transduced study subject cultures for the control of HIV that may postpone the onset of presented in this article exhibited a significant selective AIDS. advantage, although untransduced cultures never grew significantly better than the modified cultures. Since every transduced study subject culture had significant METHODS suppression of HIV replication, it is expected that a Patient recruitment and consent. Male and female HIV-infected individ- selective advantage of the modified cells should be uals age 18 and older with viral loads above 500 copies/ml and a CD4 observed in comparison to untransduced cells. The count greater than 200 cells/mm3 who were not currently on HAART strength and length of the HIV challenge in this study were included for study. Study subjects were not considered if they had was insufficient to measure selection in every patient any opportunistic infections. Human samples for our studies were obtained from The Gary Lambert Research Center at The Johns Hopkins culture. This is supported by the observation that cul- University after an IRB-approved informed consent notifying the study tures having the highest levels of p24, reflecting virion subject that the blood would be used for research purposes (J patients) production, in the untransduced cultures each demon- was obtained. Subject U01 was obtained from the University of Penn- strated a selective advantage in their transduced coun- sylvania as part of a blood distribution dry run for the phase I clinical terparts, while those with low levels of virion trial. Subject confidentiality was maintained by the use of a code known only to the principal investigator. production did not. One exception to this is patient J32, who had a low level of virion production but whose Cell isolation, culture, transduction, and challenge. Peripheral blood transduced cells still showed a significant growth advan- from healthy donors was obtained from AllCells (Berkeley, CA, USA), tage. These observations suggest that selective out- and uninfected or HIV+ study subject PBMCs were isolated by Ficoll – growth of resistant cells in vivo could vary between Hypaque density gradient separation. Study subjects numbered J23 and higher and U01 had their blood collected in CPT tubes (Becton – patients following autologous T cell therapy with Dickinson, Franklin Lakes, NJ, USA), and the cells were isolated by VRX496-modified cells. Therefore, some patients may centrifugation at 2600 rpm for 30 min at room temperature, then benefit from additional selection of vector-modified plasma was removed and cells were resuspended in blocking buffer cells, for example by incorporating a drug-resistance (PBS Ca2 Mg2, 0.5% BSA, 5 mM EDTA, and purified human immu- + gene such as MGMT into the vector and then selecting noglobulin). CD4 T lymphocytes were subsequently purified by posi- tive selection by MACS (Miltenyi Biotec, Auburn, CA, USA). Purity was for those cells in vivo [35,36]. determined to be above 95% by flow cytometry. In an ongoing clinical trial [25], inclusion criteria CD4+ T lymphocytes isolated from healthy donors were cultured in X- allow selection of only those patients who have failed Vivo-15 (BioWhittaker) containing 10% human serum (NABI, Boca Raton, several conventional treatment options and who there- FL, USA). For transduction, plates were precoated with Retronectin (Takara Bio, Inc., Japan) at a concentration of 1.5 Ag/cm2, and VRX494 fore have few alternative options for therapy. This trial is vector was loaded at 20 TU per cell onto the plates 30 min prior to bead designed to establish the safety of the vector in terms of and cell addition. T lymphocytes were plated at 1 106 cells per well in a generation of a RCL and any serious adverse event relat- 24-well plate concomitant with immobilized CD3/28 beads, using anti- ing to the dosing that results in a 0.5 log increase in viral human CD3 (clone BB11; Diaclone, France) and anti-human CD28 (clone load or 0.5 log decrease in CD4 T cell counts. At the time BL8; Diaclone) antibodies coated on M450 epoxy beads (Dynal, Lake Success, NY, USA) at a ratio of 3 beads per cell and 100 U/ml IL-2 (Chiron, of this publication, there are no safety concerns, and Emeryville, CA, USA). The method of T cell expansion is described in three patients have been dosed with no occurrence of further detail in Refs. [37,38]. For study subject cultures to which vector adverse events. Additional secondary endpoints include was added on day 2, fresh vector was added to the medium without measurement of vector mobilization and persistence of additional culture manipulation. T lymphocytes were cultured for 3 days, vector-modified cells. We have shown in vitro and in a centrifuged and washed three times to remove vector, and replated in 2 ml of medium, and 4 days later the beads were removed. If necessary, 2 mouse model that VRX496 mobilizes very little, if at all additional milliliters of medium was added between removal of vector [21]. This is most likely due to the presence of antisense and bead removal.

MOLECULAR THERAPY Vol. 9, No. 6, June 2004 911 Copyright B The American Society of Gene Therapy ARTICLE doi:10.1016/j.ymthe.2004.03.005

HIVNL4-3 (X4) was purchased from ABL, Inc. (Kensington, MD, USA). The patient studies were supported by an SBIR Phase I small business grant from HIV strains IIIB (X4), JRFL (R5), Ba-L (R5), 89.6 (X4/R5), and 93US151 (X4/ the National Institutes of Health, R43 AI51908-01. R5) were obtained from the Center for AIDS Research at the University of Pennsylvania. Upon bead depletion, 2 106 transduced or untransduced RECEIVED FOR PUBLICATION DECEMBER 5, 2003; ACCEPTED MARCH 7, cells from healthy donors were challenged with wt-HIV at various m.o.i. 2004. overnight and then washed four times. HIV-containing supernatants were recovered twice a week and cells were counted and replated at 2 106 in 4 ml of T cell medium. REFERENCES 1. Chun, T. W., et al. (1997). Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature 387: 183 – 188. Vector. VRX494 vector is a VSV-G-pseudotyped HIV-based lentiviral 2. Siliciano, J., et al. (2003). Long-term follow-up studies confirm the stability of the latent vector containing a 937-base antisense sequence against the HIV env reservoir for HIV-1 in resting CD4+ T cells. Nat. Med. 9: 727 – 728. gene under the control of the HIV LTR. VRX494 is analogous to 3. Vigouroux, C., et al. (1999). Adverse metabolic disorders during highly active antire- VRX496, the vector being evaluated in an ongoing clinical trial, except troviral treatments (HAART) of HIV disease. Diabetes Metab. 25: 385 – 392. that it contains the entire GFP-coding region for experimental evalu- 4. Behrens, G. M. N., Stoll, M., and Schmidt, R. E. (2000). Lipodystrophy in HIV infection. What is it, what causes it, and how can it be managed? Drug Saf. 23: 57 – 76. ation in place of a GFP-derived 186-base tag region in VRX496 (Fig. 1). 5. Powderly, W. G. (2003). Long-term exposure to lifelong therapies. J. Acquired Immune In the absence of tat, GFP is expressed at lower levels by the basal Defic. Syndr. 29S: 28 – 40. activity of the HIV LTR. This activity is most likely driven from the tat- 6. Max, B., and Sherer, R. (2000). Management of the adverse effects of antiretroviral independent transcriptional complex [39]. Efficacy of the antisense is therapy and medication adherence. Clin. Infect. Dis. 30S: 96 – 116. greatly upregulated upon infection of the host cell by wt-HIV, which 7. Lucas, G. M., Chaisson, R. E., and Moore, R. D. (1999). Highly active antiretroviral provides tat and highly upregulates transcription of the anti-HIV therapy in a large urban clinic: risk factor for virologic failure and adverse drug reac- payload. tions. Ann. Intern. Med. 131: 81 – 87. Vector was produced by calcium phosphate-mediated transfection of 8. Nijuis, M., Deeks, S., and Boucher, C. (2001). Implications of antiretroviral resistance on viral fitness. Curr. Opin. Infect. Dis. 14: 23 – 28. vector plasmid with the VIRPAC packaging construct into 293 cells [40]. 9. Tuner, B. J. (1993). Adherence to antiretroviral therapy by human immunodeficiency Supernatant was collected every 12 h from 24 to 48 h after transfection, virus-infected patients. J. Infect. Dis. 185S: 145 – 151. pooled, and then concentrated by high-speed centrifugation at 10,000 g 10. 11th International Workshop for HIV Drug Resistance (2002). Rapid Report. for 12 h. The vector was resuspended at approximately 1/400 of its 11. Little, S. J., et al. (2002). Antiretroviral-drug resistance among patients recently infected original volume in a storage buffer containing 60 mM NaCl and 25 mM with HIV. N. Engl. J. Med. 347: 385 – 394. Hepes at pH 7.2 and was then frozen at 80jC. The titer of the centri- 12. Lazzarin, A., et al., TORO 2 Study Group (2003). Efficacy of enfuvirtide in patients infected fuged vector was determined by the frequency of GFP expression on HeLa- with drug-resistant HIV-1 in Europe and Australia. N. Engl. J. Med. 348: 2186 – 2195. tat cells and was found to range between 1 109 and 5 109 TU/ml. 13. Trono, D., Feinberg, M. B., and Baltimore, D. (1989). HIV-1 Gag mutants can domi- nantly interfere with the replication of the wild-type virus. Cell 59: 113 – 120. A scalable purification process was developed for VRX496 [41]. Vector 14. Sarver, N., and Rossi, J. (1993). Gene therapy: a bold direction for HIV-1 treatment. titers were determined by TaqMan PCR for copy number in HeLa-tat cells AIDS Res. Hum. Retroviruses 9: 483 – 487. and the vector was proven free of replication-competent lentivirus. Final 15. Dropulic, B., and Jeang, K.-T. (1994). Gene therapy for human immunodeficiency virus 8 9 vector titers achieved ranged between 10 and 10 TU/ml. infection: genetic antiviral strategies and targets for intervention. Hum. Gene Ther. 5: 927 – 939. 16. Veres, G., et al. (1998). Comparative analyses of intracellularly expressed antisense Measurement of p24. For measurement of intracellular p24, cells were RNAs as inhibitors of human immunodeficiency virus type 1. J. Virol. 72: 1894 – 1901. fixed in 1% paraformaldehyde and permeabilized with permeabilization 17. Qin, X.-F., An, D. S., Chen, I. S., and Baltimore, D. (2003). Inhibiting HIV-1 infection in medium ‘‘B’’ (Caltag, Burlingame, CA, USA), following the manufacturer’s human T cells by lentiviral mediated delivery of small interfering RNA against CCR5. recommendations, and then stained for p24 using the antibody RDI- Proc. Natl. Acad. Sci. USA 100: 183 – 188. conjugated KC57 (Caltag) at approximately 1 Ag/200,000 cells. Virion 18. Dropulic, B., Hermanokova, M., and Pitha, P. M. (1996). A conditionally replicating production was measured by p24 ELISA (ABL, Inc.) according to the HIV-1 vector interferes with wild type HIV-1 replication and spread. Proc. Natl. Acad. Sci. manufacturer’s specifications. USA 93: 11103 – 11108. 19. Lu, X., et al. (2004). Antisense-mediated inhibition of HIV replication using an HIV-1- based vector results in severely attenuated mutants incapable of developing resistance. GHOST cell assay. The GHOST cell assay was performed as described [30]. J. Virol. (in press). Briefly, GHOST cells containing the GFP marker gene under the control of 20. Adachi, A., et al. (1986). Production of acquired immunodeficiency syndrome-associ- the LTR and expressing the CXCR4, CCR5, X4/R5, or no coreceptors were ated retrovirus in human and nonhuman cells transfected with an infectious molecular seeded at 2 105 cells per well in a 12-well plate and cultured in the clone. J. Virol. 59: 284 – 291. appropriate selection medium of DMEM containing 10% fetal calf serum 21. Lu, X., et al. (2004). J. Gen. Med. (in press). 22. Ranga, U., et al. (1998). Enhanced T cell engraftment after retroviral delivery of an with penicillin or streptomycin. The next day, virus samples collected antiviral gene in HIV-infected individuals. Proc. Natl. Acad. Sci. USA 95: 1202 – 1206. from mock patient cultures 2 weeks post-bead removal were added to the 23. Mitsuyasu, R. T., et al. (2000). Prolonged survival and tissue trafficking following adop- cellsandculturedinthepresenceof20Ag/ml Polybrene for 4 h. tive transfer of CD4~ gene-modified autologous CD4+ and CD8+ T cells in human Afterward, medium was replaced with fresh selection medium, and cells immunodeficiency virus-infected subjects. Blood 96: 785 – 793. were incubated for 48 h. Cells were then washed and fixed with 4% 24. Walker, R. E., et al. (2000). Long-term survival of receptor-modified syngeneic T cells in paraformaldehyde in PBS for an hour. The percentage of GFP-expressing patients with human immunodeficiency virus infection. Blood 96: 467 – 474. cells was determined by FACS analysis. 25. MacGregor, R. R. (2001). Clinical protocol: a phase I open label clinical trial of the safety and tolerability of single escalating doses of autologous CD4 T cells transduced with VRX496 in HIV-positive subjects. Hum. Gene Ther. 12: 2028 – 2029. Flow cytometry. Fixed cells were costained with TC-conjugated CD4 26. Levine, B. L., et al. (1997). Effects of CD28 costimulation on long-term proliferation of (Caltag) and/or PE-conjugated antibody CD3, CD8, CD25, CD122, CD4+ T cells in the absence of exogenous feeder cells. J. Immunol. 159: 5921 – 5930. CD28, CTLA-4 (Becton – Dickinson), CD45 RA, or CD45 RO (Pharmingen) 27. Roos, M. T. L., et al. (1998). Low T-cell responses to CD3 plus CD28 monoclonal at 1 Ag/200,000 cells for 30 min and then washed once. Ten thousand antibodies are predictive of development of AIDS. AIDS 12: 1745 – 1751. events were acquired on a single-laser FACScalibur (Becton – Dickinson). 28. Hofmann, B., et al. (1987). Lymphocyte transformation response to pokeweed mitogen as a predictive marker for development of AIDS and AIDS related symptoms in homo- ACKNOWLEDGMENTS sexual men with HIV antibodies. Br. Med. J. 295: 293 – 296. 29. Schellekens, P. T., Roos, M. T., De Wolf, F., Lange, J. M., and Miedema, F. (1990). Low We thank Helen Higgins and Michelle Parrish from the Gary Lambert Research T-cell responsiveness to activation via CD3/TCR is a prognostic marker for acquired Clinic for their efforts in obtaining patient samples for this study. We also thank immunodeficiency syndrome (AIDS) in human immunodeficiency virus-1 (HIV-1) in- Lauren Korshalla, Andrew Worden, Bing Jiang, Eden Deausen, Kathy Schonely, fected men. J. Clin. Immunol. 10: 121 – 127. Karen Bengston, and Jessica Boehmer for their contributions to the manuscript. 30. Benson, R. E., Sanfridson, A., Ottinger, J. S., Doyle, C., and Cullen, B. R. (1993). Down-

912 MOLECULAR THERAPY Vol. 9, No. 6, June 2004 Copyright B The American Society of Gene Therapy doi:10.1016/j.ymthe.2004.03.005 ARTICLE

regulation of cell-surface CD4 expression by simian immunodeficiency virus nef pre- 36. Davis, B., Humeau, L., and Dropulic, B. (2004). In vivo selection for human and murine vents viral superinfection. J. Exp. Med. 177: 1561 – 1566. hematopoietic cells transduced with a therapeutic MGMT lentiviral vector that inhibits 31. Morner, A., et al. (1999). Primary human immunodeficiency virus type 2 (HIV-2) iso- HIV replication. Mol. Ther. 9: 160 – 172. lates, like HIV-1 isolates, frequently use CCR5 but show promiscuity in coreceptor 37. Levine, B.L., et al. (1996). Antiviral effect and ex vivo CD4+ T cell proliferation in HIV- usage. J. Virol. 73: 2343 – 2349. positive patients as a result of CD28 costimulation. Science 272: 1939 – 1943. 32. Aiuti, A., et al. (2002). Immune reconstitution in ADA-SCID after PBL gene therapy and 38. Levine, B. L., et al. (2002). Adoptive transfer of costimulated CD4+ T cells induces discontinuation of enzyme replacement. Nat. Med. 8: 423 – 425. expansion of peripheral T cells and decreased CCR5 expression in HIV infection. Nat. 33. Jensen, M. E., and van’t Wout, A. B. (2003). Predicting HIV-1 coreceptor usage with Med. 8: 47 – 53. sequence analysis. AIDS Rev. 5: 104 – 112. 39. Lu, X., Welsh, T. M., and Peterlin, B. M. (1992). The human immunodeficiency virus 34. Simmonds, P., Balfe, P., Peutherer, J. F., Ludlam, C. A., Bishop, J. O., and Brown, A. J. type 1 long terminal repeat specifies two different transcription complexes, only one of (1990). Human immunodeficiency virus-infected individuals contain provirus in small which is regulated by tat. J. Virol. 67: 1752 – 1760. numbers of peripheral mononuclear cells and at low copy numbers. J. Virol. 64: 864 – 872. 40. www.fda.gov/ohrms/dockets/ac/01/briefing/3794b3.htm. 35. Richard, E., et al. (2003). A bicistronic SIN-lentiviral vector containing G156A MGMT 41. Slepushkin, V., et al. (1992). Large-scale purification of a lentiviral vector by size allows selection and metabolic correction of hematopoietic protoporphyric cell lines. exclusion chromatography or mustang Q ion exchange capsule. Bioprocesses J. 2: J. Gene Med. 5: 737 – 747. 89 – 95.

MOLECULAR THERAPY Vol. 9, No. 6, June 2004 913 Copyright B The American Society of Gene Therapy