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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.

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