WO2010051521A1 - Cell therapy product for the treatment of hiv infection - Google Patents

Abstract

The invention relates to cell therapy for the treatment of HIV infection in humans. It provides a composition of genetically modified human cells for introduction into the body of a person infected with HIV, to reduce the person's viral load and to provide and reconstitute T-cells that are resistant to HIV infection. The composition comprises allogeneic or autologous human CD4+ T-cells, allogeneic or autologous human hematopoietic stem cells (HSCs), and allogeneic or autologous human mesenchymal stem cells (MSCs). Each cell comprises at least one heterologous anti-HIV sequence that inhibits HIV from infecting the cells or from replicating in the cells and at least one heterologous fate-controlling sequence. The invention also provides lentiviral vectors for making the cells, plasmids and producer cells for making the vectors, and methods for making the genetically modified cells.

Description

CELL THERAPY PRODUCT FOR THE TREATMENT OF HIV INFECTION

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS This application claims priority to U.S. Provisional Patent Application No.

61/110,411, filed October 31, 2008, which is incorporated herein by reference in its entirety.

BACKGROUND

HIV infection in humans is considered pandemic by the WHO. From 1981 to 2006, AIDS killed more than 25 million people. Approximately 40 million people worldwide are infected with HIV. At the end of 2006, an estimated 1.1 million persons in the United States were living with HIV infection, with 21% undiagnosed. It has been estimated that approximately 56,300 people were newly infected with HIV in 2006. There is still significant progression from infection to progression to AIDS, with an estimated 37,041 new AIDS cases in 2007. The total estimated number of persons living with AIDS in the U.S. and dependent areas was 468,578. In 2007, the estimated number of deaths of persons with AIDS was 14, 561.

Triple cocktail drug therapy (highly active antiretroviral therapy or HAART), once believed to have great promise in effectively combating the AIDS virus, has not proven to be the cure for AIDS. Moreover, resistance to this drug therapy is increasing; and thus it has become apparent that drug therapy is not the solution for controlling the AIDS epidemic. Additionally while there has been considerable effort to develop a vaccine against HIV, there is very little evidence of success to date. Therefore, there is a need for novel therapies. HIV/ AIDS is an ideal candidate for a novel gene therapy approach, since it is an incurable and ultimately fatal disease. Although highly active anti-retroviral therapy has significantly improved the survival of HIV-infected individuals, the duration of the response is limited by development of drug-resistant viruses, long-term toxicities, and a substantial reservoir of latently infected T-cells. The long half- life of resting memory CD4 T-cells renders HIV intrinsically incurable with the treatment regimens available today. Therefore, there is an urgent need to explore new strategies to render cells more resistant to infection and to slow or halt HIV-I replication within cells upon viral entry.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows a lentiviral vector expressing anti-CCR5, env anti-sense, and MGMT. It has a standard LTR (comprised of U3, R, and U5 regions), and contains splice donor (SD), splice acceptor sites (SA), central poly-purine tract (cPPT), and the psi packaging signal (not shown). The MGMT transgene will be driven from the native LTR, as will the shRNA construct (solid arrow), encoding the antiCCR5 shRNA sequence. In the presence of Tat, TAR- dependent LTR transcription will express the anti-HIV anti-sense (AS) RNA.

Figure 2 shows examples of seven different lentiviral vectors, two designed for hematopoietic stem cells (HCSs), two designed for T-cells, and three designed for mesenchymal stem cells (MSCs). The first two constructs transduce HSCs. They contain an anti-HIV antisense sequence (targeted to any part of HIV genome that is not in the vector), a RNAi sequence targeted to the CCR5 gene (the CCR5delta32 mutant protein could also be expressed from this construct and the region that the RNAi would bind would be made resistant to the effects of the RNAi by codon degeneration), and the MGMT fate-controlling gene, that in the presence of Temozolomide, facilitates LV-transduced HSC expansion in vivo. The second two constructs transduce T-cells. They contain an anti-HIV antisense as described above, a RNAi sequence targeted to the CCR5 gene (the CCR5delta32 mutant protein could also be expressed from this construct and the region that the RNAi would bind would be made resistant to the effects of the RNAi by codon degeneration), and two fate- controlling genes, one that functions to delete the cells (TMPK) if required (useful, for example, in allogeneic transplantation) and an shRNA targeted to the Program Cell Death gene 1 (PD-I), which facilitates expansion of antigen-stimulated T cells. (In HIV infected individuals, many T cells are targeted to HIV.) The three final constructs transduce MSCs or any substrate cell type that can be used to support the engraftment and/or expansion of HSCs. These vectors contain, in addition to any of the above combination of anti-HIV genes and fate-controlling genes, genes that promote HSCs or T cells - examples being genes or factors that facilitate engraftment, homing, survival, function and/or expansion of HSCs or T cells. Non-limiting examples of such genes are SCF, TPO, EPO, G-CSF, FLT-3L, H0X-B4.

DESCRIPTION OF THE INVENTION The invention relates to cell therapy for the treatment of HIV infection in humans. As used herein, the term "HIV" includes all clades and/or strains of human immunodeficiency virus 1 (HIV-I) and human immunodeficiency virus 2 (HIV -2). In one embodiment, the invention provides a composition of genetically modified human cells for introduction into the body of a person infected with HIV, whether or not the person is actually suffering from AIDS, to reduce the person's viral load and to provide and reconstitute T-cells that are resistant to HIV infection. The composition comprises allogeneic or autologous human CD4⁺ T-cells, allogeneic or autologous human hematopoietic stem cells (HSCs), and allogeneic or autologous human mesenchymal stem cells (MSCs). Each cell comprises at least one heterologous anti-HIV sequence that inhibits HIV from infecting the cells or from replicating in the cells and at least one heterologous fate-controlling sequence. Generally, the each cell type is either all allogeneic or all autologous, but all three cell types need not be either all allogeneic or all autologous. In one embodiment, all of the cell types are allogeneic.

The heterologous anti-HIV sequence is any DNA sequence, which has been inserted into the genome of the cells, that results in the cells being resistant to HIV infection and replication. Messenger RNA (mRNA) transcribed from the sequence, or a protein or polypeptide encoded by the sequence acts, within the cells to inhibit HIV from infecting the cells or from replicating in the cells.

In one embodiment, the DNA sequence encodes an antisense mRNA transcript that is complementary to part of the HIV genome and binds (hybridizes) to it, which inhibits the virus from replicating in the cell. As used herein, the words "encode" and "encodes" refer to the production of RNA transcripts from a DNA sequence as well as the production of polypeptides and proteins from the sequence. In one aspect of this embodiment, the antisense mRNA sequence hybridizes to all or part of the HIV gag, pol, nef, and/or env gene. In another aspect, it hybridizes to all or part of the HIV env gene. The appropriate length of the mRNA sequence can vary. In one aspect, the antisense sequence is about 500 bases to about 3000 bases long. Generally, it is about 1 kilobase to about 2 kilobases long.

In another embodiment, the DNA sequence encodes an interfering RNA sequence (RNAi). In one aspect of this embodiment, the RNAi comprises a short hairpin RNA (shRNA) embedded within a microRNA (miRNA) that down-regulates at least one of the cells' HIV receptors. In another aspect of this embodiment, the shRNA down-regulates the cells' CCR5 receptors. In still another aspect, the shRNA is embedded within another RNA sequence, preferably a miRNA. Cells process this shRNA-miRNA by enzymes, such as DROSHA and DICER, to ultimately give rise to a short interfering RNA (siRNA). The siRNA interferes with mRNA that encodes CCR5 protein, thereby down-regulating it. The heterologous fate-controlling sequence allows the cells to be selected, expanded in number, or killed. This can be achieved when contacted with certain chemicals directly in vitro or indirectly in vivo when those chemicals are administered to the human to whom the cellular composition of the invention has been administered. However, drugs are not always necessary and other methods for controlling cell fate can be accomplished by techniques known in the art. For example, the control gene can be self limiting. An example of this is the PD-I gene that is active in T-cells where the T-cell receptor (TCR) is HIV specific and during a period where there is ample HIV antigen stimulating the cells. Once HIV antigen is abated, then the PD-I gene would not be active (since it depends upon TCR signalling to facilitate cell expansion) and therefore would be self limiting. In another embodiment, the fate-controlling sequence comprises a cell -killing sequence, which encodes a protein that kills the cells when they are exposed to certain chemicals, such as certain drugs. Examples are well-known to those skilled in the art and include thymidine kinase (TK), deoxycytidine kinase (dCK), and the modified mammalian and human thymidylate kinase (TMPK) disclosed in U.S. patent application number 11/559,757, filed on November 14, 2006 and published on March 19, 2009 as US 2009/0074733 Al, which is incorporated herein by reference in its entirety. As used herein, "TMPK" refers to a nucleic acid sequence that encodes this modified human TMPK protein, "TK" refers to a nucleic acid sequence that encodes the TK protein, and "dCK" refers to a nucleic acid sequence that encodes the dCK protein. The presence of a cell-killing sequence permits the cells to be removed from the person, if necessary for safety or other reasons.

In another embodiment, the fate-controlling sequence comprises a cell selection sequence. The cell selection sequence encodes a protein that permits expansion of the genetically modified cells in vitro or in vivo by either expressing or inhibiting a cellular gene. This can be done in the presence of a pro-drug or without a pro-drug. Examples are well- known to those skilled in the art and include multidrug resistance protein 1 (MDR-I), dihydro folate reductase (DHFR), and the mutated version of methylguanine-DNA methyltransferase (also refered to as O⁶-alkylguanine-DNA alkyltransferase). This last protein and nucleic acid sequences encoding it are disclosed in U.S. Pat. No. 5,965,126, issued October 12, 1999, which is incorporated herein by reference in its entirety. As used herein, "MGMT" refers to a nucleic acid sequence that encodes this mutated human methylguanine-DNA methyltransferase. In another embodiment, the fate-controlling sequence comprises an anti-apoptosis sequence. This is a sequence that encodes a protein or a mRNA transcript, such as a RNAi, that inhibits or prevents the cell from undergoing apoptosis in circumstances where it would otherwise undergo apoptosis. In one aspect of this embodiment, the sequence encodes a shRNA that targets the programmed cell death 1 gene (PDl gene). In a further aspect, the shRNA is embedded within a miRNA.

In a further embodiment of the invention, the MSCs of the composition include at least one heterologous sequence that encodes a stem cell promotion factor. As used herein, the term "stem cell promotion factor" is any protein, polypeptide, or other molecule produced within the cells that promotes engraftment and/or proliferation of the MSCs in vitro or within the human to whom the cellular composition has been administered. In one aspect of this embodiment, the stem cell promotion factor comprises a cytokine. Cytokines are well-known to those skilled in the art, and the selection of the appropriate ones are within the skill on those in the art. Cytokines that are particularly appropriate for the MSCs of the invention include thrombopoietin (TPO), stem cell factor (SCF), erythropoietin (EPO), FLT3-ligand (FLT-3L), interleukin-3 (IL-3), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). In one particular aspect, the cytokines are SCF, TPO, FLT-3L, and/or G-CSF. In still another aspect of this embodiment, the stem cell promotion factor comprises H0X-B4. In another embodiment, either or both of the T-cells and the HSCs within the cellular composition comprise two different anti-HIV sequences that inhibit HIV from infecting those cells or from replicating in the infected cells. In one aspect, one anti-HIV sequence encodes an mRNA that is an antisense sequence that binds to at least part of the HIV genome and the other encodes an miRNA that down-regulates at least one of the cell's HIV co-receptors. As previously mentioned, the antisense sequence can hybridize to at least part of the HIV gag, pol, nef, and/or env gene, and the miRNA can be an shRNA that down-regulates the cells' CCR5 HIV co-receptors.

The cellular composition is administered to people who are infected with HIV. Thus, the invention provides a method of lowering the number of HIV particles in a human comprising administering to the human an effective amount of the composition sufficient to permit engraftment and proliferation of the genetically modified cells to sufficient levels in the body so as to dramatically impact HIV viral load and disease progression to AIDS. Since it is widely known that there is a direct correlation between viral load and disease progression, lowering the viral load will postpone the development of full-blown AIDS, possibly indefinitely. The hope of this therapy is to decrease the HIV viral load to levels that are not conducive to the development of AIDS.

As previously mentioned, the composition comprises three types of modified human cells: CD4⁺ T-cells, HSCs, and MSCs. Each type of cell has been genetically engineered to include and express the DNA sequences discussed above. The therapy can use one, two, all three, or any combination of the types of genetically modified cells described herein.

The allogeneic or autologous human CD4+ T-cell comprises at least one heterologous anti-HIV sequence that inhibits HIV from infecting the cell or from replicating in the cell, and it comprises at least one heterologous fate-controlling sequence. In one embodiment, the cell comprises two different heterologous anti-HIV sequences. In a further embodiment, it also comprises two different heterologous fate-controlling sequences.

The T-cells are obtained from human blood. Autologous cells are obtained from the blood of the same person to whom the modified T-cells will be administered. Allogeneic cells are obtained from a genetically different person. Generally, standard techniques in the art are used to obtain allogeneic cells that best match the tissue type of the intended recipient.

The anti-HIV sequence can be a sequence that encodes an antisense RNA sequence that binds to at least part of the HIV genome or a sequence that encodes an shRNA that down-regulates at least one of the cell's HIV co-receptors. In one aspect, the antisense sequence binds to at least part of the HIV gag, pol, nef , and/or env genes. In a further aspect, it binds to at least part of the HIV env gene. In a preferred aspect, the antisense sequence is about 500 bases to about 3000 bases long and typically about one kb to about two kb long. In one aspect, a shRNA that is embedded within a miRNA down-regulates the cell's HIV CCR5 co-receptors. In one embodiment, the heterologous fate-controlling sequence is selected from the group consisting of a cell-killing sequence and an anti-apoptosis sequence. In one aspect, the cell-killing sequence comprises TK or dCK. In another aspect, it comprises TMPK. In one aspect, the anti-apoptosis sequence comprises a sequence that encodes a shRNA that targets the PDl gene. In a further aspect, the shRNA is embedded within another RNA sequence to enhance the processing of the shRNA. Optionally, this sequence is an miRNA.

Like the T-cell, the allogeneic or autologous human HSC comprises at least one heterologous anti-HIV sequence that inhibits HIV from infecting the cell or from replicating in the cell, and it comprises at least one heterologous fate-controlling sequence. In one embodiment, the cell comprises two different heterologous anti-HIV sequences. In a further embodiment, it also comprises two different heterologous fate-controlling sequences. The anti-HIV sequences are the ones described above.

The HSCs are obtained from human bone marrow, human blood, human umbilical cord, or human umbilical cord blood. Generally, allogeneic cells are matched to the tissue type of the intended recipient.

In one embodiment, the heterologous fate-controlling sequence is a cell-killing sequence or a cell selection sequence. The cell-killing sequence is as described for the T-cell. In one embodiment, the cell selection sequence is a sequence encoding neomycin resistance protein, MDRl, or mutant DHFR. In one aspect of this embodiment, the cell selection sequence comprises MGMT.

Like the T-cell and the HSC, the allogeneic or autologous human MSC comprises at least one heterologous anti-HIV sequence that inhibits HIV from infecting the cell or from replicating in the cell and at least one heterologous fate-controlling sequence. In one aspect of this embodiment, the anti-HIV sequence comprises the various anti-sense sequences described above. In another aspect of this embodiment, the fate-controlling sequence comprises a cell-killing sequence as described above.

The MSCs are obtained from various human tissues, including bone marrow, blood, umbilical cord, and umbilical cord blood. Generally, allogeneic cells are matched to the tissue type of the intended recipient.

In one embodiment, the MSC further comprises a sequence that encodes a stem cell promotion factor. In one aspect of this embodiment, the stem cell promotion factor comprises a cytokine. In a further aspect, the cell can include one or more heterologous sequences encoding one or more of the following cytokines: TPO, SCF, EPO, FLT-3L, IL-3, G-CSF, and GM-CSF. In a still further aspect, the cytokine comprises one or more of SCF, TPO, FLT-3L, and G-CSF. In another aspect of this embodiment, the stem cell promotion factor comprises H0X-B4.

All three of the modified cell types may be made by various techniques known to those skilled in the art. In one embodiment, they are made by transducing the unmodified cells with lentiviral vectors to introduce the desired sequences into the genomes of the cells.

The lentiviral vector of the invention comprises at least one heterologous anti-HIV sequence that inhibits HIV from infecting a cell that it otherwise could infect or from replicating in an infected cell, and it comprises at least one heterologous fate-controlling sequence.

In one embodiment, the anti-HIV sequence comprises an antisense sequence to at least part of the HIV genome. In one aspect of this embodiment, the antisense sequence binds to at least part of the HIV env, gag, pol, and/or nef gene. In a further aspect, the antisense sequence is about 500 bases to about 3000 bases long. Generally, it is about one kb to two kb long. In a still further aspect, the antisense sequence binds to at least part of the

HIV env gene.

In another embodiment, the anti-HIV sequence encodes an shRNA that down- regulates at least one of the cell's HIV receptors. In one aspect of this embodiment, the receptor is CCR5. In another aspect, the shRNA is embedded within another sequence, which is optionally a miRNA.

In another embodiment, the heterologous fate-controlling sequence is one or more of the following: 1) a cell-killing sequence, 2) a cell selection sequence, 3) an anti-apoptosis sequence, and 4) a sequence encoding a stem cell promotion factor. In one aspect, the cell- killing sequence comprises TK, dCK, or TMPK. In a preferred aspect, it comprises TMPK.

In one aspect, the cell selection sequence comprises a sequence encoding MDR-I, neomycin resistance protein, and DHFR. In a preferred aspect, it comprises MGMT. In one aspect, the anti-apoptosis sequence comprises a sequence that encodes an shRNA that targets the PDl gene. In a preferred aspect, the shRNA is embedded within a miRNA. In one aspect, the sequence encoding a stem cell promotion factor encodes one or more cytokines. In one aspect, the cytokine is one or more of TPO, SCF, EPO, FLT-3L, IL-3, G-CSF, and GM-CSF.

In a preferred aspect, the cytokine is one or more of SCF, TPO, FLT-3L, and G-CSF. In still another aspect, the stem cell promotion factor comprises HOX-B4. Certain vectors of the invention are particularly useful for transducing the particular types of cells that comprise the therapeutic cellular composition. These vectors and their cell types are shown in Figure 2.

For HSCs, the first vector comprises: 1) an anti-HIV sequence that encodes an antisense RNA sequence that binds to any part of the HIV genome that is not in the vector, for example, to at least part of the HIV gag, pol, nef, or env gene, 2) an anti-HIV sequence that encodes an miRNA embedded shRNA that down-regulates the CCR5 HIV co-receptors on the surface of a cell that can be infected by HIV, and 3) a cell selection sequence comprising MGMT. The second vector comprises the sequences of the first vector plus a cell-killing sequence comprising TMPK.

For T-cells, the first vector comprises: 1) an anti-HIV sequence that encodes an antisense RNA sequence that binds to any part of the HIV genome that is not in the vector, for example, to at least part of the HIV gag, pol, nef, or env gene, 2) a cell-killing sequence that comprises TMPK, and 3) an anti-apoptosis sequence comprising a sequence that encodes an miPvNA embedded shRNA that targets the PD-I gene. The second vector comprises the sequences of the first vector plus an anti-HIV sequence that encodes a shRNA that down- regulates the CCR5 co-receptors on the surface of a cell that can be infected by HIV. For MSCs, the first vector comprises: 1) an anti-HIV sequence that encodes an antisense RNA sequence that binds to any part of the HIV genome that is not in the vector, for example, to at least part of the HIV gag, pol, nef, or env gene, 2) a cell-killing sequence that comprises TMPK, and 3) sequences encoding the cytokines TPO, SCF, and FLT-3L. The second vector comprises the antisense sequence and the cell-killing sequence of the first vector plus a sequence encoding G-CSF. The third vector comprises the antisense sequence and the cell-killing sequence of the first vector plus a sequence encoding H0X-B4.

The lentiviral vectors are constructed by techniques known to those skilled in the art. Such techniques are disclosed in U.S. Patent Application No. 11/884,639, published as US 2008/0254008 Al, and in U.S. Patent Nos. 5,994,136, 6,013,516, 6,165,782, 6,294,165 Bl, 6,428,953 Bl, 6,797,512 Bl, 6,863,884 B2, 6,924,144 B2, 7,083,981 B2, and 7,250,299 Bl, the disclosures of which are incorporated herein by reference in their entireties. Generally, plasmids are prepared that contain one or more of the heterologous sequences disclosed herein. The plasmids are transfected into packaging cells that contain an HIV gag-pol sequence and a sequence that encodes a pseudo-typed envelope protein, such as the vesicular stomatitis virus protein G (VSV-G). This creates producer cells that produce the desired lentiviral vectors. These cells are mammalian cells, generally human cells. In one aspect of the invention, they are human embryonic kidney cells, such as HEK 293 cells. The producer cells are cultured under standard conditions for such cells, and the vectors are recovered from the supernatant. Thus, the plasmid of the invention comprises at least one heterologous anti-HIV sequence that inhibits HIV from infecting a cell that it otherwise could infect or from replicating in an infected cell, and it comprises at least one heterologous fate-controlling sequence. The sequences are those described above with respect to the lentiviral vectors. The plasmids that produce the vectors shown in Figure 2 contain the same sequences. The sequences can all be in one plasmid or they can be in multiple plasmids.

The following examples illustrate certain aspects of the invention and should not be construed as limiting the scope thereof.

EXAMPLES Background

Lentiviral vector LG896 is designed to render the immune system free of the pathologic effects of HIV infection by the expression of three distinct transcripts in genetically modified CD4 progenitors by transducing CD34+ hematopoietic stem cells

(HSC). Two of the transcripts serve an anti-HIV function and the third, P 140K MGMT, will provide in vivo selection of transduced HSC. The first anti-HIV transcript encoded by the LV will serve to decrease the infectability of target cells by down-modulating the CCR5 co- receptor for HIV. CCR5 blockade on its own has shown great potential for protecting cells from infection [I]. The second anti-HIV transcript is a 1 kb anti-sense sequence to the HIV env sequence. This transcript will limit productive HIV infection in instances where CCR5 blockade (encoded by the first transcript) was overcome. The therapeutic env anti-sense transcript will rapidly associate with the single-stranded RNA genome of the virus and any mRNA env transcripts. Once bound, the double-stranded RNA will be rapidly degraded by the host cell. The use of a long transcript 1 kb sequence helps to avoid problems with sequence variability [2].

A second cell, the CD34+ hematopoietic stem cell (HSC), will also be transduced. Lentiviral vectors (LV) are ideally suited for the transduction of HSC given the permanency of expression of the integrated transgene and the superior safety profile of LV over onco- retroviral vectors. While HSC can be harvested from mobilized peripheral blood, successful long-term engraftment of gene modified autologous cells requires "space" to be created in the bone marrow. This normally requires full lymphodepletion (as occurs during bone marrow transplantation, HSCT). That is why MGMT is incorporated into the LV. By expressing the MGMT gene in HSC, short courses of Temozolomide chemotherapy can be given to deplete native HSC and allow gene-modified HSC to expand and permanently engraft. If successful, this would insure that a permanent supply of HIV-resistant lymphocytes would be generated by the patient's own hematopoietic system. While the ability to select for MGMT-expressing HSC upon treatment with alkylating agents such as temozolomide has been proven in numerous animal models (simian, canine, murine), the demonstration of this technology in a clinical trial has yet to occur and would significantly advance the field of gene therapy as a whole - as numerous other disorders, such as inherited monogenic defects, could also be corrected using this technology [3][4][5][6][7][8]. The primary hypothesis is that Lentiviral vector expression of two anti-HIV transcripts in CD4 T cells that are derived from in vivo selected LG896 modified HSCs will result in a decreased viral load and stabilization of patient CD4 cells counts by interfering with the HIV replication cycle a proportionally large number of genetically modified CD4 T cells in vivo. It is well established that there is a direct correlation between viral load and AIDS disease progression. By establishing a large number of CD4 T cells in the body by the infusion of LG896 CD4 T cells and HSCs after transplantation and selection with Temozolomide, it is hypothesized that this will significantly decrease viral load to levels that will significantly impact disease progression.

HIV/ AIDS is an ideal candidate for a novel gene therapy approach since it is an incurable and terminal disease. Although highly active anti-retroviral therapy has significantly improved the survival of HIV-infected individuals, the duration of response is limited by development of drug-resistant viruses, long-term toxicities and a substantial reservoir of latently infected T-cells [9] [1O][I I]. Tremendous advances have been made over the past several years in understanding HIV pathogenesis. Following primary infection, the virus replicates in local lymph nodes, then disseminates in a massive viremia. Although HIV- 1 elicits a strong immune response in most infected individuals, the virus almost invariably escapes immune containment [12]. Persistent infection is characterized by the gradual decrease of CD4+ T cells, ultimately leading to AIDS. In the vector to be constructed, Tat will drive the expression of the anti-en v transcript. In the absence of Tat, the splice donor and acceptor sites present in the vector backbone will allow expression of downstream MGMT and shRNA sequences (for anti-CCR5). The shRNA sequence specific for CCR5 will be expressed in the context of a micro-RNA sequence. This miRNA expression system for shRNA is the basis for the SMART vector™ program marketed by Thermo Scientific. Diminished cell surface CCR5 expression, caused by anti-CCR5 shRNA expression, will result in diminished HIV infectability of the target cell. If infected, Tat-mediated expression of the antisense transcript will render the infection in that cell non-productive. The third element encoded by the vector, MGMT, is a well-described tool for expanding the percentage of transduced HSC in the marrow of treated subjects. Subsequently, the LG896 vector will be tested in clinical trials involving HIV/ AIDS patients and will significantly challenge existing therapies, which are currently inadequate, for providing stable and prolonged therapeutic activity.

HIV-I infection is characterized by a host- virus relationship in which the virus utilizes the host cell's macromolecular machinery and energy supplies to produce progeny virus[14]. Eventually, HIV-I alters the cell's physiological state, leading to disruption of immune responses and cell death. Specific viral and cellular proteins are known to play crucial roles in the alteration of cellular functions. Examples include the HIV-I accessory proteins and host cell chemokine coreceptors, CCR5 and CXCR4, which are essential for HIV- 1 infection [ 16] [ 17] . Host cell target genes such as Ets- 1 , CDK4, NFAT 1 and NFAT 1 [18] [19] [20] [21] induce enhanced HIV-I expression in vitro, although information on the role of each of these gene products in in vivo infection is limited. The HIV-I accessory proteins (Tat, Rev, Nef, Vif, Vpu and Vpr) are regulatory proteins encoded by HIV-I, which are involved in HIV infectivity, transcription, replication and/or pathogenesis [22] [23]. Functional inhibitors of these viral regulatory proteins are not currently available.

Today, the only proven therapy against HIV-I in vivo is a cocktail of anti-retroviral drugs (HAART), comprised of two reverse transcriptase inhibitors and a protease inhibitor. This therapy has led to maintenance of very low viral load in the majority of treated patients [24]. This therapy is not, however, able to induce sustained suppression or cure. Even if the plasma viral level falls below the current level of detection, HIV-I continues to replicate at very low levels or persist in a reservoir of latently infected T cells [25]. Therapies in the experimental stages of testing include vaccines based on engineered gpl20-CD4-CCR5 fusion proteins, which have been shown to elicit antibodies capable of neutralizing infectivity of primary HIV-I isolates [26]. However, evidence of in vivo efficacy is not yet available. Therefore, there is an urgent need to explore novel strategies that slow or halt HIV-I replication, reactivation, and reinfection of naϊve T cells. Infection with HIV-I is associated with a progressive decrease in CD4+ T cell number and a consequent impairment in host immune defenses. Generating a population of T cells that are resistant to viral infection and to virus-induced pathology if cells are infected would be ideally suited to counter this inexorable march of the virus through the immune system.

Latently infected resting CD4 T cells provide the major reservoir for HIV in individuals on highly active antiretro viral therapy (HAART) [27]. Two distinct forms of latency are thought to occur. 1) Pre-integration latency involves recently infected resting CD4 T cells that harbor partially or completely reverse transcribed HIV DNA in a labile pre- integration complex [28]. Although pre -integration latency is the most prevalent form of latency in untreated HIV infection, it is highly labile, decaying with a half- life of 1-6 days. If these cells are activated before the pre -integration complex becomes non-functional, then nuclear import, integration, and productive infection will occur [29][30][28]. 2) Post- integration latency is observed in resting CD4 T cells, typically in memory cells that have a copy of the HIV genome stably integrated into chromosomal DNA [31] [32]. Activation of these cells leads to viral gene expression and replication. The long half-life of these resting memory CD4 T cells renders HIV intrinsically incurable with the treatment regimens currently available [33].

Eliminating latently infected CD4 T cells: Reactivation of pre- and post- integration latency has been ttempted in vitro by stimuli that trigger T cell activation (i.e. antibodies to CD3 orthe PMA homologue prostatin, or specifϊccytokines) [34][35][36][37][38]. Control and, ultimately, cure of HIV infection requires a therapy either kills the latently infected cell or, disrupts the ability to infect and replicate in the naϊve CD4 T cell population. Killing ofat least some latently infected cells has been achieved in vitro by an anti-CD45RO immunotoxin, although levels of CD45RO are too low for detection on many infected cells [39] [40]. Disrupting HIV replication in the naϊve CD4T cell population could significantly impact the disease since this population drives the generation of high viral loads and therefore progression to full-blown AIDS. While non-CD4 T cells harboring latent HIV genomes are sources for future infection of naϊve CD4 T cells, they do not themselves generate the high viral load sneeded to drive disease progression. Therefore, it is hypothesized that selection of LG896 HSCs with Temozolomide in vivo would generate a high percentage of LG896 modified CD4 T cells that would significantly decrease the viral load to levels that are not conducive to the development of full-blown AIDS.

Lentiviral vectors (LV) provide highly efficient and stable gene expression. Lentivirus gene therapy vectors promise to be safer than the murine oncoretroviral-based (MLV) vectors recently used in the successful treatment of disease in three gene therapy trials to date, including an X-linked SCID trial [65][66][67]. Contrary to the MLV used in these trials, LV are not associated with oncogenesis and therefore may represent a safety advantage over oncoretroviral gene therapy vectors [68][69][70][71][72][73]. In particular, LV are thought not to have the same enhancer activity as oncoretrovirus-based vectors. The long terminal repeat (LTR) has a low basal rate of activity and expression is driven by Tat binding to the TAR element in the LTR to facilitate elongation of HIV RNA, rather than the upregulation of transcriptional activity via enhancer-DNA binding protein complexes. Furthermore, HIV- based LV have less poly- A read through into neighboring genes [74]. The reduced risk of lentivirus mediated insertional oncogenesis is underscored by the fact that leukemia is not a recognized side effect of HIV patients and is extremely rare, even though memory T- cells are known to harbor integrated virus for years and many of these pro viruses are defective and therefore do not kill the host cell [75][32][76]. Notably, none of the patients in the recently finished Phase I clinical trial of the HIV-based LV have experienced any adverse events due to treatment, thus supporting the safety of LV vectors for applications in humans [13]. Research Design and Methods

To generate LG896, a LV expressing: a) a miRNA that down regulates CCR5 by expression of a CCR5-specific shRNA, b) a 1 kb anti-sense transcript to the HIV viral genome, and c) the MGMT gene in Lentigen's LentiMax system.

To initiate studies that lead to a new therapeutic vector, LG896, for HIV/ AIDS, the design, molecular cloning, and validation of a new vector will be carried out. The hypothesis is that this vector will effectively produce therapeutic CCR5 -specific and MGMT transcripts in non-infected cells and only produce anti-HIV antisense transcripts in HIV-infected cells. To construct LV that expresses an anti-CCR5 shRNA in the context of miRNA, the P140K MGMT gene, and a 1 kb anti-sense env transcript from a Tat-inducible native LTR.

Rationale: The lentiviral vector genome, as developed into a gene delivery system by Lentigen, can be manipulated in a number of ways in order to express new RNAs or proteins in vector-treated host cells. The LentiMax system should efficiently express antisense RNA for env, the MGMT gene and shRNA for the knock-down of CCR5 from the vector shown schematically in Figure 1. The advantage of this vector is that different amounts of the three RNA species will be expressed depending upon whether or not the target cell becomes infected with HIV. In non-infected cells there will be a significant level of CCR5- specific shRNA and MGMT transcripts formed from the activity of the native LTR promoter. Transcripts from the native LTR will initiate in the LTR, then utilize the splice donor and acceptor sites, (SA, SD) resulting in the transcription of MGMT and miRNA from backbone sequence that follows the SA sequence. If a transduced cell becomes infected with HIV, the Tat transcriptional regulatory protein and Rev post-transcriptional regulatory proteins will also be expressed. Tat will bind to the TAR element, which upregulates transcription and, in concert with nuclear-exporter Rev, expresses the anti-sense transcript (that is located between the splice donor (SD) and splice acceptor (SA) sites) for export and targeting of wt-HIV genomic RNA. In toto, CCR5-specifϊc shRNA and MGMT are expressed prior to prevent HIV infection and generate HIV-resistant cells. However, if the virus breakthrough occurs, then the long lkb anti-HIV antisense is expressed in Tat and Rev dependent manner to prevent productive HIV replication. The long antisense is not expressed constitutively prior to HIV infection, but only after HIV breakthrough infection, needing the in-coming wt-HIV s Tat and Rev to express the anti-HIV antisense sequence, target wt-HIV RNA and interfere with productive HIV replication. Experimental Plan: Starting with the parental LentiMax plasmid that utilizes a native, as opposed to SIN LTRs, an anti-sense sequence for env from the HIV-1NL4-3 molecular clone, immediately after the slice donor and before the rev-response element (RRE), will be cloned. The P 140K MGMT gene will be cloned immediately following the splice acceptor (SA) site. Immediately following the cDNA for P 140K MGMT, a sequence encoding a shRNA for CCR5, expressed in the context of a miRNA, will be inserted. The sequence will be prepared by Dharmacon's (now a part of ThermoFisher Scientific). To titer and perform quality control of generated vector.

Rationale: Once the vector is produced by transient transfection of a 293 HEK producer cell line, the harvested vector-containing supernatant will be titered and the infectious titer, without the production of replication competent lentivirus (RCL), demonstrated.

Experimental Plan: The vector backbone produced in Aim Cl .A. will be used to transduce 293 cells (by calcium phosphate transduction, along with packaging and VSV-G envelope constructs) for the purpose of producing infectious lentiviral vector particles (LV). For initial laboratory studies, supernatant will be collected 24 and 48 hours post transfection, pooled and concentrated by high-speed centrifugation at 9,500 rpm for 14 hours. Vector will be resuspended in approximately 1/500 of its original volume in a vector storage buffer (65 mM NaCl and 20 mM HEPES) at pH 7.22, and frozen at -80 C.

Dilutions of the harvested supernatant will then be used to infect new cultures of HEK 293 cells in order to determine lentiviral vector titer. Titer will be determined by real-time PCR. A 100 bp specific DNA sequence is part of the Lentigen vector system that was introduced into the vector specifically for quantitation. The 5' forward and 3' reverse primers that will be used are: 5'-CCACTCCTGACAACTACTCT, and 5'- GGAGTTGAGACCAGTGTAGT. The real-time PCR probe is 5'- CAGTAGGTGAAGGAGTCGTAGTTG. The genomic DNA of transduced cells will be isolated using the DNAeasy kit (Qiagen) and real-time PCR performed on normalized (for DNA concentration) samples using primers and probe. Typical titers range between 1-7 xl O⁹ transducing units (TU) per ml. The titer will then be used to determine the amount of vector needed to transduce a high proportion of target cells in subsequent studies.

One primary concern for retrovirus-based gene delivery vectors is the development of RCL, replication competent retrovirus. In order to test for RCL following production, the concentrated lentiviral vector preparation will be tested on C8166-45 indicator cells. An attenuated HIV unable to express the HIV-I accessory proteins vif, vpr, vpu, and nef will be used as the positive control. This will be an appropriate control because none of these HIV-I accessory genes are expressed in the vector or packaging constructs, and therefore would not be present in RCL generated during vector production. This attenuated control will be measured for TCID50 on the C8166 indicator cell line. Real-time PCR using a specific TaqMan probe will be used as one method to measure the positive control and putative RCL in the supernatant of the indicator cell lines. Specifically, HIV gag and VSV-G RNA will be analyzed after passage of the vector on the indicator cell line. The assay will also be used to detect residual VSV-G DNA in the vector prep. We will also measure p24. P24 production in the supernatant of C8166 cells will be measured by p24 ELISA (ABL, Inc.) according to manufacturer's specifications.

For sterility and mycoplasma, tests will be performed as follows: 10 ul of concentrated vector will be added to 10 ml LB broth and grown for 72 hours at 37⁰C. The culture is then examined for turbidity and mycoplasma testing is performed by PCT using a mycoplasma detection kit (Sigma Aldrich) according to the manufacturer's instructions. The basal state of expression of the three transgenes will be determined by real-time

PCR in the vector as a quality measure. Although subsequent aims will examine expression under different conditions, the expression of transgenes in HEK 293 cells would be the approach that is most likely to meet with regulatory agency approval. To optimize protocols for transduction of primary T cells and HSC with LG896. The ability to reliably infect primary T cells and CD34+ hematopoietic stem cells will be demonstrated with the LG896 vector.

Experimental Plan: Human CD34+ hematopoietic stem cells will be obtained from left over cord blood samples Primary human T cells will be obtained from healthy volunteers. Human CD34 will be cultured for 2 days in growth factors in vitro and transduced on each of two consecutive days with a range of MOI (1 to 75) in triplicate wells. On day 3, HSC will then be cultured in methylcellulose (MethoCult, StemCEll Technologiers, Vancouver, BC, with hemin, SCF, IL-3, GMCSF and epo [82]) and assayed for colony progenitor (HPC) cell activity. The assessment of HPC activity is standard, as is the quantification of percent transduction of HSC by RT-PCR for MGMT by plucking individual colonies from culture 13 days after plating. Extensive historical data exists for a vector Lentigen has produced expressing MGMT (LG631), and thus comparison to the single MGMT vector will serve as a positive control. For the transduction of primary human T cells the following methodology will be used. Human PBMC will stimulated with rIL-2, and anti- CD3/anti-CD28 beads (Dynabeads ClinExVivo CD3/CD28 in Optimizer media, as per Invitrogen), in retronectin-coated cell processing bags in the presence of LV-containing supernatant [13]. The three parameters that will be tested are: a) the ability of transduced lymphocytes to expand in vitro upon subsequent re-stimulation, and b) the expression of each vector-encoded RNA in the transduced cell population as determined by RT-PCR, and c) the expression of CCR5 on the surface of transduced lymphocytes as determined by flow cytometry.

To demonstrate the ability of the triple-anti-HIV vector (LG896) to transduce lymphocytes and render them resistant to HIV infection. To determine the expression level of each transcript of the triple vector in transduced lymphocyte cell lines expressing CCR5, macrophages, and T cells by RT-PCR in naive cells, cells mock-infected following transduction, and HIV-I infected cells following vector transduction.

Rationale: the expression of RNA encoding anti-sense env and anti-CCR5 shRNA will be tested in two immortalized cell lines that express CCR5, monocyte-derived macrophages, and primary T cells. Documented expression of LV-encoded transgenes will justify testing the construct for the ability to prevent HIV infection in future clinical studies.

Experimental Plan: Suptl/CCR5 T cells and PMl cells (NIH-ARRRP) are immortalized cell lines that express CCR5 and are cultured in standard media. Monocytes will be purified from normal human volunteers by CDl 1 b+ cell sorting and then differentiated into macrophages with GM-CSF and M-CSF as described by Oberling et al., [83][84]. CD4 cells will be expanded from density gradient purified lymphocytes using anti- CD3/anti-CD28 beads (as above). Expression of CCR5 on the cell surface, as well as antibody for phenotypic characterization (CD3, CD4, CDl 1 b,CD68) will be determined by flow cytometry (BD Pharmingen), as described in Cordelier, etal [42]. Each cell type will be transduced with LG896 at an MOI of 50 for two consecutive days, cultured for 3-4 days, and then stained for surface expression of CCR5. GFP expressing vector will serve as a positive control for transduction of each cell type, as well as a test for non-specific CCR5 down- regulation. HIV-I infection assays will be carried out initially with HIV-I Ba-L (0.05-1.5 ng p24 equivalents of cell free virus) but then with other strains (NL4-3; X4 strain etc). After overnight incubation, cells will be washed and every 2-3 days thereafter and assayed for expression of phenotypic markers. At each sub-culture step, 100,000 infected and non- infected cells will be harvested for quantification of LV-transduction levels (% transduction), HIV-I infection (of integrated genomes) by DNA PCR, and for LG896 encoded RNA for MGMT, anti-sense env, and shRNA for CCR5 by RT-PCR. The transduction level over time will be determined by RT-PCR wherein copies of integrated DNA are correlated to the overall genome number. To quantify the amount of virus produced from naive, mock-infected, and HIV-I infected cells.

Rationale: Cell types tested in C.2. A., with and without prior transduction with LG896, will be tested for their ability to support HIV-I infection.

Experimental Plan: After overnight incubation with HIV-I (initially the Ba-L strain, representing a R5 tropic strain), cells will be washed and every 2-3 days thereafter supernatants assayed for p24 levels by ELISA (ZeptoMetrix). The transduction level over time will be determined by RT-PCR wherein copies of integrated DNA are correlated to the overall genome number, as described above. At each wash step, phenotypic markers and CCR5 surface expression will be analyzed by flow cytometry. To quantify the viability of transduced cells following exposure to HIV-I.

Rationale: In addition to the inhibition of HIV-I, the vector the vector will be tested for untoward effects on cell viability.

Experimental Plan: T lymphocytes will be activated with anti-CD3/anti-CD28 beads and transduced with vector on days 0 and 1. Viability will be assessed by flow cytometric analysis by Annexin-V and 7- AAD staining every 2-3 days along with phenotypic staining for CD3, CD4, and CCR5.

The impact on cell viability for CD34+ HSC will be analyzed in two ways. First, at the end of the two day infection cycle, viability of control vs. LG896 transduced CD34+ HSCs (HPCs) will be determined by trypan blue exclusion. Comparison will be made between the total number of HSCs that grow out in in vitro culture.

To transduce cord blood CD34+ HSC with LG896, adoptively transfer transduced HSC to NOD/SCID mice, and assess engraftment with and without pharmacologic selection by virtue of MGMT expression.

The model for in vivo function of transduced cells will be the re-population of NOD/SCID mice with transduced CD34+ HSC. The NOD/SCID model is appropriate as human CD34+ HSC engraft and generate CFU that can be subsequently measured in vitro. Moreover, the function of the MGMT gene can be readily assessed by the amplification of the percent transduced cells upon administration of BCNU to the mice.

To demonstrate the ability to infect CD34+ HSC with LG896, and to determine the impact, if any, on CFU formation ability.

Rationale: Prior to in vivo assessment OΪLG896 transduced CD34+HSC, it will have been demonstrated that transduction does not affect CFU- forming ability in vitro. Transduction efficiencies in the 50%-90% range are anticipated.

Experimental Plan: Human umbilical cord derived CD34+ cells will be cultured, transduced with LG896 at an MOI of 50 for two consecutive days, and then transferred to NOD/SCID mice as described in previous work [82] [60]. Prior to transfer, a subset of these cells will be cultured in methylcellulose for CFU, in the presence or absence of BCNU. Up to 20 colonies will be picked and PCR analyzed for transgene expression.

To demonstrate the ability of LG896 transduced HSC to repopulate NOD/SCID mice, and to increase the proportion of transduced cells in the marrow by MGMT-mediated drug selection.

Rationale: The presence of the MGMT gene in the LG896 vector should allow for the in vivo selection of transduced cells by treatment with BCNU. Having demonstrated that LG896 does not negatively impact NOD/SCID repopulating cells in vitro, the ability to specifically select for and expand MGMT-expressing cells in vivo will be demonstrated.

Experimental Plan: Human CD34 cells will be transduced with LG896 as in C.3. A. These cells will then be mixed with non-transduced cells from the same donor to create a cell population that has the number OΪLG896 transduced cells decreased by either 50% or by

90%. Next, an equivalent number of control, 100% exposed to LG896, 50%, or 10% will be infused into NOD/SCID mice. Mice will then be treated with BCNU and the percentage of LG896 transduced cells in the bone marrow demonstrated by harvesting the marrow from experimental animals, expanding CFU progenitors in vitro in methylcellulose culture, and determining the percentage of colonies that are positive for the vector. Control MGMT vector, LG631, which has been experimentally demonstrated to expand human CD34 cells in NOD/SCID mice treated with BCNU, will be used as a positive control.

REFERENCES

1 Baba M, Nishimura O, Kanzaki N, Okamoto M, Sawada H, Iizawa Y, Shiraishi M, Aramaki Y, Okonogi K, Ogawa Y, Meguro K, Fujino M: A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity. Proc Natl Acad Sci U S A 1999, 96:5698-5703.

2 Lu X, Yu Q, Binder GK, Chen Z, Slepushkina T, Rossi J, Dropulic B: Antisense- mediated inhibition of human immunodeficiency virus (HIV) replication by use of an HIV type 1 -based vector results in severely attenuated mutants incapable of developing resistance. J Virol 2004, 78:7079-7088. 3 Persons DA, Allay ER, Sawai N, Hargrove PW, Brent TP, Hanawa H, Nienhuis AW,

Sorrentino BP: Successful treatment of murine beta-thalassemia using in vivo selection of genetically modified, drug- resistant hematopoietic stem cells. Blood 2003, 102:506-513.

4 Neff T, Horn PA, Peterson LJ, Thomasson BM, Thompson J, Williams DA, Schmidt M, Georges GE, von Kalle C, Kiem H: Methylguanine methyltransferase-mediated in vivo selection and chemoprotection of allogeneic stem cells in a large-animal model. J Clin Invest 2003, 112:1581-1588.

5 Davis BM, Koc ON, Gerson SL: Limiting numbers of G156A O(6)-methylguanine- DNA methyltransferase-transduced marrow progenitors repopulate nonmyeloablated mice after drug selection. Blood 2000, 95:3078-3084. 6 Beard BC, Sud R, Keyser KA, Ironside C, Neff T, Gerull S, Trobridge GD, Kiem H:

Long-term polyclonal and multilineage engraftment of MGMTP 140K gene -modified dog hematopoietic cells in primary and secondary recipients. Blood 2009, :.

7 Davis BM, Humeau L, Dropulic B: In vivo selection for human and murine hematopoietic cells transduced with a therapeutic MGMT lentiviral vector that inhibits HIV replication. MoI Ther 2004, 9:160- 172.

8 Neff T, Beard BC, Peterson LJ, Anandakumar P, Thompson J, Kiem H: Polyclonal chemoprotection against temozolomide in a large-animal model of drug resistance gene therapy. Blood 2005, 105:997- 1002. 9 Vigouroux C, Gharakhanian S, Salhi Y, Nguyen TH, Chevenne D, Capeau J, Rozenbaum W: Diabetes, insulin resistance and dyslipidaemia in lipodystrophic HIV-infected patients on highly active antiretroviral therapy (HAART). Diabetes Metab 1999, 25:225-232.

10 Sedaghat AR, Siliciano RF, Wilke CO: Low-level HIV-I replication and the dynamics of the resting CD4+ T cell reservoir for HIV-I in the setting of HAART. BMC Infect Dis 2008, 8:2.

11 Sedaghat AR, Dinoso JB, Shen L, Wilke CO, Siliciano RF: Decay dynamics of HIV-I depend on the inhibited stages of the viral life cycle. Proc Natl Acad Sci U S A 2008, 105:4832-4837. 12 Goulder P, Price D, Nowak M, Rowland- Jones S, Phillips R, McMichael A: Co- evolution of human immunodeficiency virus and cytotoxic T-lymphocyte responses. Immunol Rev 1997, 159:17-29.

13 Levine BL, Humeau LM, Boyer J, MacGregor R, Rebello T, Lu X, Binder GK, Slepushkin V, Lemiale F, Mascola JR, Bushman FD, Dropulic B, June CH: Gene transfer in humans using a conditionally replicating lentiviral vector. Proc Natl Acad Sci U S A 2006, 103:17372-1 7377.

14 Fauci AS: Host factors and the pathogenesis of HIV -induced disease. Nature 1996, 384:529-534.

15 Pantaleo G, Fauci AS: Immunopathogenesis of HIV infection. Annu Rev Microbiol 1996, 50:825-854.

16 Liu R, Paxton WA, Choe S, Ceradini D, Martin SR, Horuk R, MacDonald ME, Stuhlmann H, Koup RA, Landau NR: Homozygous defect in HIV-I coreceptor accounts for resistance of some multiply-exposed individuals to HIV-I infection. Cell 1996, 86:367-377.

17 Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, Di Marzio P, Marmon S, Sutton RE, Hill CM, Davis CB, Peiper SC, Schall TJ, Littman DR, Landau NR:

Identification of a major co-receptor for primary isolates of HIV-I. Nature 1996, 381 :661- 666.

18 Posada R, Pettoello-Mantovani M, Sieweke M, Graf T, Goldstein H: Suppression of HIV type 1 replication by a dominant-negative Ets-1 mutant. AIDS Res Hum Retro viruses 2000, 16:1981-1989.

19 Nekhai S, Shukla RR, Fernandez A, Kumar A, Lamb NJ: Cell cycle-dependent stimulation of the HIV-I promoter by Tat-associated CAK activator. Virology 2000, 266:246-256. 20 Cron RQ, Bartz SR, Clausell A, Bort SJ, Klebanoff SJ, Lewis DB: NFATl enhances HIV-I gene expression in primary human CD4 T cells. Clin Immunol 2000, 94:179-191.

21 Kinoshita S, Su L, Amano M, Timmerman LA, Kaneshima H, Nolan GP: The T cell activation factor NF-ATc positively regulates HIV-I replication and gene expression in T cells. Immunity 1997, 6:235-244.

22 Emerman M, Malim MH: HIV-I regulatory/accessory genes: keys to unraveling viral and host cell biology. Science 1998, 280:1880-1884.

23 Casella CR, Rapaport EL, Finkel TH: Vpu increases susceptibility of human immunodeficiency virus type 1-infected cells to fas killing. J Virol 1999, 73:92-100. 24 Bushman F, Landau NR, Emini EA: New developments in the biology and treatment of HIV. Proc Natl Acad Sci U S A 1998, 95:11041-11042.

25 Chun TW, Stuyver L, Mizell SB, Ehler LA, Mican JA, Baseler M, Lloyd AL, Nowak

MA, Fauci AS: Presence of an inducible HIV-I latent reservoir during highly active antiretroviral therapy. Proc Natl Acad Sci U S A 1997, 94:13193-13197. 26 LaCasse RA, Follis KE, Trahey M, Scarborough JD, Littman DR, Nunberg JH:

Fusion-competent vaccines: broad neutralization of primary isolates of HIV. Science 1999,

283:357-362.

27 Blankson JN, Persaud D, Siliciano RF: The challenge of viral reservoirs in HIV-I infection. Annu Rev Med 2002, 53:557-593. 28 Zack JA, Arrigo SJ, Weitsman SR, Go AS, Haislip A, Chen IS: HIV-I entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell

1990, 61 :213-222.

29 Spina CA, Guatelli JC, Richman DD: Establishment of a stable, inducible form of human immunodeficiency virus type 1 DNA in quiescent CD4 lymphocytes in vitro. J Virol 1995, 69:2977-2988.

30 Bukrinsky MI, Stanwick TL, Dempsey MP, Stevenson M: Quiescent T lymphocytes as an inducible virus reservoir in HIV-I infection. Science 1991, 254:423-427.

31 Chun TW, Finzi D, Margolick J, Chadwick K, Schwartz D, Siliciano RF: In vivo fate of HIV- 1-infected T cells: quantitative analysis of the transition to stable latency. Nat Med 1995, 1 :1284-1290.

32 Chun TW, Carruth L, Finzi D, Shen X, DiGiuseppe JA, Taylor H, Hermankova M, Chadwick K, Margolick J, Quinn TC, Kuo YH, Brookmeyer R, Zeiger MA, Barditch-Crovo P, Siliciano RF: Quantification of latent tissue reservoirs and total body viral load in HIV-I infection. Nature 1997, 387:183-188.

33 Finzi D, Blankson J, Siliciano JD, Margolick JB, Chadwick K, Pierson T, Smith K, Lisziewicz J, Lori F, Flexner C, Quinn TC, Chaisson RE, Rosenberg E, Walker B, Gange S, Gallant J, Siliciano RF: Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-I, even in patients on effective combination therapy. Nat Med 1999, 5:512-517.

34 Korin YD, Brooks DG, Brown S, Korotzer A, Zack JA: Effects of prostratin on T-cell activation and human immunodeficiency virus latency. J Virol 2002, 76:8118-8123. 35 Kulkosky J, Culnan DM, Roman J, Dornadula G, Schnell M, Boyd MR, Pomerantz RJ: Prostratin: activation of latent HIV-I expression suggests a potential inductive adjuvant therapy for HAART. Blood 2001, 98:3006-3015.

36 Brooks DG, Hamer DH, Arlen PA, Gao L, Bristol G, Kitchen CMR, Berger EA, Zack JA: Molecular characterization, reactivation, and depletion of latent HIV. Immunity 2003, 19:413-423.

37 Brooks DG, Arlen PA, Gao L, Kitchen CMR, Zack JA: Identification of T cell- signaling pathways that stimulate latent HIV in primary cells. Proc Natl Acad Sci U S A 2003, 100:12955-12960.

38 Scripture -Adams DD, Brooks DG, Korin YD, Zack JA: Interleukin-7 induces expression of latent human immunodeficiency virus type 1 with minimal effects on T-cell phenotype. J Virol 2002, 76:13077- 13082.

39 McCoig C, Van Dyke G, Chou CS, Picker LJ, Ramilo O, Vitetta ES: An anti- CD45RO immunotoxin eliminates T cells latently infected with HIV-I in vitro. Proc Natl Acad Sci U S A 1999, 96:11482-11485. 40 Saavedra-Lozano J, McCoig C, Xu J, Cao Y, Keiser P, Ghetie V, Siliciano RF,

Siliciano JD, Picker LJ, Ramilo O, Vitetta ES: An anti-CD45RO immunotoxin kills latently infected human immunodeficiency virus (HIV) CD4 T cells in the blood of HIV-positive persons. J Infect Dis 2002, 185:306-314.

41 Dropulic B, June CH: Gene-based immunotherapy for human immunodeficiency virus infection and acquired immunodeficiency syndrome. Hum Gene Ther 2006, 17:577-

588.

42 Cordelier P, Kulkowsky JW, Ko C, Matskevitch AA, McKee HJ, Rossi JJ, Bouhamdan M, Pomerantz RJ, Kari G, Strayer DS: Protecting from R5-tropic HIV: individual and combined effectiveness of a hammerhead ribozyme and a single-chain Fv antibody that targets CCR5. Gene Ther 2004, 11:1627- 1637.

43 Hϋtter G, Nowak D, Mossner M, Ganepola S, Mϋssig A, Allers K, Schneider T, Hofmann J, Kϋcherer C, Blau O, Blau IW, Hofmann WK, Thiel E: Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. N Engl J Med 2009, 360:692-698.

44 Olson WC, Jacobson JM: CCR5 monoclonal antibodies for HIV-I therapy. Curr Opin HIV AIDS 2009, 4:104-111.

45 Moore JP, Kuritzkes DR: A piece de resistance: how HIV-I escapes small molecule CCR5 inhibitors. Curr Opin HIV AIDS 2009, 4:118-124. 46 Agrawal L, Maxwell CR, Peters PJ, Clapham PR, Liu SM, Mackay CR, Strayer DS: Complexity in human immunodeficiency virus type 1 (HIV-I) co-receptor usage: roles of CCR3 and CCR5 in HIV-I infection of monocyte-derived macrophages and brain microglia. J Gen Virol 2009, 90:710-722.

47 Cordelier P, Morse B, Strayer DS: Targeting CCR5 with siRNAs: using recombinant SV40-derived vectors to protect macrophages and microglia from R5 -tropic HIV.

Oligonucleotides 2003, 13:281-294.

48 Banerjea A, Li M, Remling L, Rossi J, Akkina R: Lentiviral transduction of Tar Decoy and CCR5 ribozyme into CD34+ progenitor cells and derivation of HIV-I resistant T cells and macrophages. AIDS Res Ther 2004, 1 :2. 49 Anderson J, Li M, Palmer B, Remling L, Li S, Yam P, Yee J, Rossi J, Zaia J, Akkina R: Safety and efficacy of a lentiviral vector containing three anti-HIV genes— CCR5 ribozyme, tat-rev siRNA, and TAR decoy—in SCID-hu mouse-derived T cells. MoI Ther 2007, 15:1182-1188.

50 Kohn DB, Bauer G, Rice CR, Rothschild JC, Carbonaro DA, Valdez P, Hao QL, Zhou C, Bahner I, Kearns K, Brody K, Fox S, Haden E, Wilson K, Salata C, Dolan C, Wetter C, Aguilar-Cordova E, Church J: A clinical trial of retro viral-mediated transfer of a rev- responsive element decoy gene into CD34(+) cells from the bone marrow of human immunodeficiency virus- 1 -infected children. Blood 1999, 94:368-371.

51 Amado RG, Mitsuyasu RT, Rosenblatt JD, Ngok FK, Bakker A, Cole S, Chorn N, Lin L, Bristol G, Boyd MP, MacPherson JL, Fanning GC, Todd AV, Ely JA, Zack JA, Symonds

GP: Anti-human immunodeficiency virus hematopoietic progenitor cell-delivered ribozyme in a phase I study: myeloid and lymphoid reconstitution in human immunodeficiency virus type- 1 -infected patients. Hum Gene Ther 2004, 15:251- 262. 52 Podsakoff GM, Engel BC, Carbonaro DA, Choi C, Smogorzewska EM, Bauer G, Selander D, Csik S, Wilson K, Betts MR, Koup RA, Nabel GJ, Bishop K, King S, Schmidt M, von Kalle C, Church JA, Kohn DB: Selective survival of peripheral blood lymphocytes in children with HIV-I following delivery of an anti- HIV gene to bone marrow CD34(+) cells. MoI Ther 2005, 12:77-86.

53 Karran P, Macpherson P, Ceccotti S, Dogliotti E, Griffin S, Bignami M: 06- methylguanine residues elicit DNA repair synthesis by human cell extracts. J Biol Chem 1993, 268:15878-15886.

54 Xu-Welliver M, Kanugula S, Pegg AE: Isolation of human O6-alkylguanine-DNA alkyltransferase mutants highly resistant to inactivation by O6-benzylguanine. Cancer Res

1998, 58:1936-1945.

55 Crone TM, Goodtzova K, Edara S, Pegg AE: Mutations in human O6-alkylguanine- DNA alkyltransferase imparting resistance to O6-benzylguanine. Cancer Res 1994, 54:6221- 6227. 56 Reese JS, Ko? ON, Lee KM, Liu L, Allay JA, Phillips WPJ, Gerson SL: Retroviral transduction of a mutant methylguanine DNA methyltransferase gene into human CD34 cells confers resistance to 06- benzylguanine plus 1, 3 -bis(2-chloroethyl)-l -nitrosourea. Proc Natl Acad Sci U S A 1996, 93:14088-14093.

57 Reese JS, Davis BM, Liu L, Gerson SL: Simultaneous protection of G156A methylguanine DNA methyltransferase gene-transduced hematopoietic progenitors and sensitization of tumor cells using O6-benzylguanine and temozolomide. Clin Cancer Res 1999, 5:163-169.

58 Ragg S, Xu-Welliver M, Bailey J, D'Souza M, Cooper R, Chandra S, Seshadri R, Pegg AE, Williams DA: Direct reversal of DNA damage by mutant methyltransferase protein protects mice against dose- intensified chemotherapy and leads to in vivo selection of hematopoietic stem cells. Cancer Res 2000, 60:5187-5195.

59 Ko? ON, Reese JS, Davis BM, Liu L, Majczenko KJ, Gerson SL: DeltaMGMT- transduced bone marrow infusion increases tolerance to O6-benzylguanine and l,3-bis(2- chloroethyl)-l -nitrosourea and allows intensive therapy of l,3-bis(2-chloroethyl)-l- nitrosourea-resistant human colon cancer xenografts. Hum Gene Ther 1999, 10:1021-1030.

60 Zielske SP, Reese JS, Lingas KT, Donze JR, Gerson SL: In vivo selection of MGMT(P 140K) lentivirus-transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning. J Clin Invest 2003, 112:1561-1570. 61 Sawai N, Zhou S, Vanin EF, Houghton P, Brent TP, Sorrentino BP: Protection and in vivo selection of hematopoietic stem cells using temozolomide, O6-benzylguanine, and an alkyltransferase-'expressing retroviral vector. MoI Ther 2001, 3:78-87.

62 Neff T, Horn PA, Valli VE, Gown AM, Wardwell S, Wood BL, von Kalle C, Schmidt M, Peterson LJ, Morris JC, Richard RE, Clackson T, Kiem H, Blau CA: Pharmacologically regulated in vivo selection in a large animal. Blood 2002, 100:2026-2031.

63 Richard E, Robert E, Cario-Andre M, Ged C, Geronimi F, Gerson SL, de Verneuil H, Moreau-Gaudry F: Hematopoietic stem cell gene therapy of murine protoporphyria by methylguanine-DNA-methyltransferase-mediated in vivo drug selection. Gene Ther 2004, 11 :1638-1647.

64 Quinn JA, Desjardins A, Weingart J, Brem H, Dolan ME, Delaney SM, Vredenburgh J, Rich J, Friedman AH, Reardon DA, Sampson JH, Pegg AE, Moschel RC, Birch R, McLendon RE, Provenzale JM, Gururangan S, Dancey JE, Maxwell J, Tourt-Uhlig S, Herndon JE2, Bigner DD, Friedman HS: Phase I trial of temozolomide plus 06- benzylguanine for patients with recurrent or progressive malignant glioma. J Clin Oncol 2005, 23:7178-7187.

65 Hacein-Bey-Abina S, Garrigue A, Wang GP, Soulier J, Lim A, Morillon E, Clappier E, Caccavelli L, Delabesse E, Beldjord K, Asnafϊ V, Maclntyre E, Dal Cortivo L, Radford I, Brousse N, Sigaux F, Moshous D, Hauer J, Borkhardt A, Belohradsky BH, Wintergerst U, Velez MC, Leiva L, Sorensen R, Wulffraat N, Blanche S, Bushman FD, Fischer A,

Cavazzana-Calvo M: Insertional oncogenesis in 4 patients after retro virus-mediated gene therapy of SCID-Xl. J Clin Invest 2008, 118:3132-3142.

66 Blaese RM, Culver KW, Miller AD, Carter CS, Fleisher T, Clerici M, Shearer G, Chang L, Chiang Y, Tolstoshev P, Greenblatt JJ, Rosenberg SA, Klein H, Berger M, Mullen CA, Ramsey WJ, Muul L, Morgan RA, Anderson WF: T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years. Science 1995, 270:475-480.

67 Aiuti A, Slavin S, Aker M, Ficara F, Deola S, Mortellaro A, Morecki S, Andolfi G, Tabucchi A, Carlucci F, Marinello E, Cattaneo F, Vai S, Servida P, Miniero R, Roncarolo MG, Bordignon C: Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 2002, 296:2410-2413.

68 Wu X, Li Y, Crise B, Burgess SM: Transcription start regions in the human genome are favored targets for MLV integration. Science 2003, 300:1749-1751. 69 Schroder ARW, Shinn P, Chen H, Berry C, Ecker JR, Bushman F: HIV-I integration in the human genome favors active genes and local hotspots. Cell 2002, 110:521-529.

70 Mitchell RS, Beitzel BF, Schroder ARW, Shinn P, Chen H, Berry CC, Ecker JR, Bushman FD: Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences. PLoS Biol 2004, 2:E234.

71 Kaiser J: Gene therapy. Seeking the cause of induced leukemias in X-SCID trial. Science 2003, 299:495.

72 Hematti P, Hong B, Ferguson C, Adler R, Hanawa H, Sellers S, Holt IE, Eckfeldt CE, Sharma Y, Schmidt M, von Kalle C, Persons DA, Billings EM, Verfaillie CM, Nienhuis AW, Wolfsberg TG, Dunbar CE, Calmels B: Distinct genomic integration of MLV and SIV vectors in primate hematopoietic stem and progenitor cells. PLoS Biol 2004, 2:e423.

73 De Palma M, Montini E, Santoni de Sio FR, Benedicenti F, Gentile A, Medico E, Naldini L: Promoter trapping reveals significant differences in integration site selection between MLV and HIV vectors in primary hematopoietic cells. Blood 2005, 105:2307-2315. 74 Zaiss A, Son S, Chang L: RNA 3' readthrough of oncoretro virus and lentivirus: implications for vector safety and efficacy. J Virol 2002, 76:7209-7219. 75 Herndier BG, Shiramizu BT, Jewett NE, Aldape KD, Reyes GR, McGrath MS: Acquired immunodeficiency syndrome-associated T-cell lymphoma: evidence for human immunodeficiency virus type 1-associated T-cell transformation. Blood 1992, 79:1768-1774. 76 Baum C, Dϋllmann J, Li Z, Fehse B, Meyer J, Williams DA, von Kalle C: Side effects of retroviral gene transfer into hematopoietic stem cells. Blood 2003, 101 :2099-2114.

77 Rossi JJ: Dotting the I's and crossing the T's: integration analyses in transduced patient T cells. MoI Ther 2009, 17:756-757.

78 Wang GP, Levine BL, Binder GK, Berry CC, Malani N, McGarrity G, Tebas P, June CH, Bushman FD: Analysis of lentiviral vector integration in HIV+ study subjects receiving autologous infusions of gene modified CD4+ T cells. MoI Ther 2009, 17:844-850.

79 Lu X, Humeau L, Slepushkin V, Binder G, Yu Q, Slepushkina T, Chen Z, Merling R, Davis B, Chang Y, Dropulic B: Safe two-plasmid production for the first clinical lentivirus vector that achieves >99% transduction in primary cells using a one-step protocol. J Gene Med 2004, 6:963-973.

80 Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, Royal RE, Topalian SL, Kammula US, Restifo NP, Zheng Z, Nahvi A, de Vries CR, Rogers-Freezer LJ, Mavroukakis SA, Rosenberg SA: Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 2006, 314:126-129.

81 Zielske SP, Lingas KT, Li Y, Gerson SL: Limited lentiviral transgene expression with increasing copy number in an MGMT selection model: lack of copy number selection by drug treatment. MoI Ther 2004, 9:923-931.

82 Zielske SP, Gerson SL: Lentiviral transduction of P 140K MGMT into human CD34(+) hematopoietic progenitors at low multiplicity of infection confers significant resistance to BG/BCNU and allows selection in vitro. MoI Ther 2002, 5:381-387.

83 Vincent F, Eischen A, Bergerat JP, Faradji A, Bohbot A, Oberling F: Human blood- derived macrophages: differentiation in vitro of a large quantity of cells in serum- free medium. Exp Hematol 1992, 20:17-23.

84 Eischen A, Vincent F, Bergerat JP, Louis B, Faradji A, Bohbot A, Oberling F: Long term cultures of human monocytes in vitro. Impact of GM-CSF on survival and differentiation. J Immunol Methods 1991, 143:209-221. All publications, including issued patents and published applications, and all database entries identified by url addresses or accession numbers are incorporated herein by reference in their entirety.

Although this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

Claims

WHAT IS CLAIMED IS:

1. A lentiviral vector comprising at least one heterologous anti-HIV sequence that inhibits HIV from infecting a cell that it otherwise could infect or from replicating in an infected cell and further comprising at least one heterologous fate-controlling sequence.

2. The vector of claim 1 wherein one of the anti-HIV sequences encodes an antisense RNA sequence that binds to at least part of the HIV genome.

3. The vector of claim 2 wherein the antisense RNA sequence binds to at least part of the HIV env, gag, pol or nef gene.

4. The vector of claim 3 wherein the antisense sequence is about 500 bases to about

3000 bases long.

5. The vector of claim 4 wherein the antisense sequence is about one kb to two kb long.

6. The vector of claim 4 wherein the antisense sequence binds to at least part of the HIV env gene.

7. The vector of claim 6 further comprising a second anti-HIV sequence that encodes an shRNA that down-regulates at least one of the cell's HIV receptors.

8. The vector of claim 7 wherein the shRNA is embedded within another sequence, optionally a miRNA.

9. The vector of claim 7 wherein the receptor is CCR5.

10. The vector of claim 1 wherein the fate-controlling sequence comprises a cell-killing sequence.

11. The vector of claim 10 wherein the cell-killing sequence comprises TK or dCK.

12. The vector of claim 10 wherein the cell-killing sequence comprises TMPK.

13. The vector of claim 1 wherein the fate-controlling sequence comprises a cell selection sequence.

14. The vector of claim 13 wherein the cell selection sequence is selected from the group consisting of sequences encoding MDR-I, DHFR, and neomycin resistance protein.

15. The vector of claim 13 wherein the cell selection sequence comprises MGMT.

16. The vector of claim 1 wherein the fate-controlling sequence comprises an anti- apoptosis sequence.

17. The vector of claim 16 wherein the anti-apoptosis sequence comprises a sequence that encodes an shRNA that targets the PDl gene.

18. The vector of claim 17 wherein the shRNA is embedded within a miRNA.

19. The vector of claim 7 further comprising at least one sequence encoding a stem cell promotion factor.

20. The vector of claim 19 wherein the stem cell promotion factor comprises a cytokine.

21. The vector of claim 20 wherein the cytokine is selected from the group consisting of TPO, SCF, EPO, FLT-3L, IL-3, G-CSF, and GM-CSF.

22. The vector of claim 20 wherein the cytokine comprises SCF, TPO, FLT-3L, or G- CSF.

23. The vector of claim 19 wherein the stem cell promotion factor comprises H0X-B4.

24. The vector of claim 21 wherein the fate-controlling sequence comprises TMPK.

25. A lentiviral vector comprising an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV gag, pol, nef, or env gene, an anti-HIV sequence that encodes an miRNA embedded shRNA that down-regulates the CCR5 HIV co-receptors on the surface of a cell that can be infected by HIV, and a cell selection sequence comprising MGMT.

26. The lentiviral vector of claim 25 further comprising a cell-killing sequence comprising TMPK.

27. A lentiviral vector comprising an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV gag, pol, nef, or env gene, a cell-killing sequence comprising TMPK, and an anti-apoptosis sequence comprising a sequence that encodes an miRNA embedded shRNA that targets the PD-I gene.

28. The lentiviral vector of claim 27 further comprising an anti-HIV sequence that encodes a shRNA that down-regulates the CCR5 co-receptors on the surface of a cell that can be infected by HIV.

29. A lentiviral vector comprising an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV gag, pol, nef, or env gene, a cell-killing sequence comprising TMPK, and a sequence encoding the cytokines TPO, SCF, and FLT-3L.

30. A lentiviral vector comprising an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV gag, pol, nef, or env gene, a cell-killing sequence comprising TMPK, and a sequence encoding G-CSF.

31. A lentiviral vector comprising an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV gag, pol, nef, or env gene, a cell-killing sequence comprising TMPK, and a sequence encoding H0X-B4.

32. The lentiviral vector of any one of claims 1-31 wherein the anti-HIV sequences are anti-HIV-1 sequences.

33. A plasmid comprising at least one heterologous anti-HIV sequence that inhibits HIV from infecting a cell that it otherwise could infect or from replicating in an infected cell and further comprising at least one heterologous fate-controlling sequence.

34. The plasmid of claim 33 wherein the anti-HIV sequence comprises an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV genome or a sequence that encodes an shRNA that down-regulates at least one of the cell's HIV receptors.

35. The plasmid of claim 34 wherein the antisense sequence binds to at least part of the

HIV gag, pol, nef ,or env genes.

36. The plasmid of claim 35 wherein the antisense sequence is about 500 bases to about 3000 bases long.

37. The plasmid of claim 36 wherein the antisense sequence is about one kb to 2 kb long.

38. The plasmid of claim 36 wherein the antisense sequence binds to at least part of the

HIV env gene.

39. The plasmid of claim 34 wherein the shRNA down-regulates the CCR5 co-receptor.

40. The plasmid of claim 33 wherein the heterologous fate-controlling sequence is selected from the group consisting of a cell-killing sequence, a cell selection sequence, and an anti-apoptosis sequence.

41. The plasmid of claim 40 wherein the cell-killing sequence comprises TK or dCK.

42. The plasmid of claim 40 wherein the cell-killing sequence comprises TMPK.

43. The plasmid of claim 40 wherein the cell selection sequence is selected from the group consisting of sequences encoding neomycin resistance protein, MDRl, and DHFR.

44. The plasmid of claim 40 wherein the cell selection sequence comprises MGMT.

45. The plasmid of claim 40 wherein the anti-apoptosis sequence comprises a sequence that encodes an shRNA that targets the PDl gene.

46. The plasmid of claim 45 wherein the shRNA is embedded within a miRNA.

47. The plasmid of claim 33 comprising two different heterologous anti-HIV sequences.

48. The plasmid of claim 47 wherein the anti-HIV sequences comprise an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV genome and a sequence that encodes a shRNA that down-regulates at least one of the cell's CCR5 HIV co-receptors and expresses a codon modified CCR5delta32 protein that is not targeted by the shRNA.

49. The plasmid of claim 48 wherein the antisense sequence binds to at least part of the

HIV gag, pol nef, or env gene and the shRNA is embedded within a miRNA.

50. The plasmid of claim 49 wherein the antisense sequence binds to at least part of the

HIV env gene.

51. The plasmid of claim 33 comprising two different heterologous fate-controlling sequences.

52. The plasmid of claim 51 wherein the fate-controlling sequences are selected from the group consisting of a cell-killing sequence, a cell selection sequence, and an anti- apoptosis sequence.

53. The plasmid of claim 52 wherein the cell-killing sequence comprises TMPK, the cell selection sequence comprises MGMT, and the anti-apoptosis sequence comprises a sequence that encodes an miRNA embedded shRNA that targets the PDl gene.

54. The plasmid of claim 33 further comprising at least one nucleic acid sequence encoding a stem cell promotion factor.

55. The plasmid of claim 54 wherein the stem cell promotion factor comprises a cytokine.

56. The plasmid of claim 55 wherein the cytokine is selected from the group consisting of TPO, SCF, EPO, FLT-3L, IL-3, G-CSF, and GM-CSF.

57. The plasmid of claim 55 wherein the cytokine comprises SCF, TPO, FLT-3L, or G-

CSF.

58. The plasmid of claim 54 wherein the stem cell promotion factor comprises H0X-B4.

59. A plasmid comprising an antisense sequence that binds to at least part of the HIV gag, pol, nef, or env gene, anti-HIV sequence that encodes a shRNA that down-regulates the CCR5 HIV co-receptors on the surface of a cell that can be infected by HIV, and a cell selection sequence comprising MGMT.

60. The plasmid of claim 59 further comprising a cell-killing sequence comprising TMPK.

61. A plasmid comprising an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV gag, pol, nef, or env gene, a cell-killing sequence comprising TMPK, and an anti-apoptosis sequence comprising a sequence that encodes an shRNA that targets the PDl gene.

62. The plasmid of claim 61 further comprising an anti-HIV sequence that encodes an miRNA that down-regulates the CCR5 co-receptors on the surface of a cell that can be infected by HIV.

63. A plasmid comprising an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV gag, pol, nef, or env gene, a cell-killing sequence comprising TMPK, and a sequence encoding the cytokines TPO, SCF, and FLT-3L.

64. A plasmid comprising an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV gag, pol, nef, or env gene, a cell-killing sequence comprising TMPK, and a sequence encoding G-CSF.

65. A plasmid comprising an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV gag, pol, nef, or env gene, a cell-killing sequence comprising TMPK, and a sequence encoding H0X-B4.

66. The plasmid of any one of claims 33-65 wherein the anti-HIV sequences are anti- HIV- 1 sequences.

67. A producer cell comprising the plasmid of any one of claims 33-65.

68. The producer cell of claim 67 wherein the cell is a mammalian cell.

69. The producer cell of claim 68 wherein the mammalian cell is a human cell.

70. The producer cell of claim 69 wherein the human cell is a human embryonic kidney cell, optionally a HEK 293 cell.

71. Lentiviral vectors produced by the producer cell of claim 67.

72. Lentiviral vectors produced by the producer cell of claim 68.

73. Lentiviral vectors produced by the producer cell of claim 69.

74. Lentiviral vectors produced by the producer cell of claim 70.

75. A producer cell comprising the plasmid of claim 66.

76. The producer cell of claim 75 wherein the cell is a mammalian cell.

77. The producer cell of claim 76 wherein the mammalian cell is a human cell.

78. The producer cell of claim 77 wherein the human cell is a human embryonic kidney cell, optionally a HEK 293 cell.

79. Lentiviral vectors produced by the producer cell of claim 75.

80. Lentiviral vectors produced by the producer cell of claim 76.

81. Lentiviral vectors produced by the producer cell of claim 77.

82. Lentiviral vectors produced by the producer cell of claim 78.

83. A producer cell comprising the vector of any one of claims 1-31.

84. The producer cell of claim 83 wherein the cell is a mammalian cell.

85. The producer cell of claim 84 wherein the mammalian cell is a human cell.

86. The producer cell of claim 85 wherein the human cell is a human embryonic kidney cell, optionally a HEK 293 cell.

87. A producer cell comprising the vector of claim 32.

88. The producer cell of claim 87 wherein the cell is a mammalian cell.

89. The producer cell of claim 88 wherein the mammalian cell is a human cell.

90. The producer cell of claim 89 wherein the human cell is a human embryonic kidney cell, optionally a HEK 293 cell.

91. A method of producing lentiviral vectors comprising the steps of culturing the producer cell of claim 67 and recovering the vectors produced by the cells.

92. A method of producing lentiviral vectors comprising the steps of culturing the producer cell of claim 68 and recovering the vectors produced by the cells.

93. A method of producing lentiviral vectors comprising the steps of culturing the producer cell of claim 69 and recovering the vectors produced by the cells.

94. A method of producing lentiviral vectors comprising the steps of culturing the producer cell of claim 70 and recovering the vectors produced by the cells.

95. A method of producing lentiviral vectors comprising the steps of culturing the producer cell of claim 75 and recovering the vectors produced by the cells.

96. A method of producing lentiviral vectors comprising the steps of culturing the producer cell of claim 76 and recovering the vectors produced by the cells.

97. A method of producing lentiviral vectors comprising the steps of culturing the producer cell of claim 77 and recovering the vectors produced by the cells.

98. A method of producing lentiviral vectors comprising the steps of culturing the producer cell of claim 78 and recovering the vectors produced by the cells.

99. A method of producing lentiviral vectors comprising the steps of culturing the producer cell of claim 83 and recovering the vectors produced by the cells.

100. A method of producing lentiviral vectors comprising the steps of culturing the producer cell of claim 84 and recovering the vectors produced by the cells.

101. A method of producing lentiviral vectors comprising the steps of culturing the producer cell of claim 85 and recovering the vectors produced by the cells.

102. A method of producing lentiviral vectors comprising the steps of culturing the producer cell of claim 86 and recovering the vectors produced by the cells.

103. A method of producing lentiviral vectors comprising the steps of culturing the producer cell of claim 87 and recovering the vectors produced by the cells.

104. A method of producing lentiviral vectors comprising the steps of culturing the producer cell of claim 88 and recovering the vectors produced by the cells.

105. A method of producing lentiviral vectors comprising the steps of culturing the producer cell of claim 89 and recovering the vectors produced by the cells.

106. A method of producing lentiviral vectors comprising the steps of culturing the producer cell of claim 90 and recovering the vectors produced by the cells.

107. An allogeneic or autologous human CD4⁺ T-cell comprising at least one heterologous anti-HIV sequence that inhibits HIV from infecting the cell or from replicating in the cell and further comprising at least one heterologous fate-controlling sequence.

108. The T-cell of claim 107 wherein the anti-HIV sequence comprises a sequence that encodes an antisense RNA sequence that binds to at least part of the HIV genome or a sequence that encodes an shRNA embedded within a miRNA that down-regulates at least one of the cell's HIV co-receptors.

109. The T-cell of claim 108 wherein the antisense sequence binds to at least part of the HIV gag, pol, nef , or env gene.

110. The T-cell of claim 109 wherein the antisense sequence is about 500 bases to about 3000 bases long.

111. The T-cell of claim 110 wherein the antisense sequence is about one kb to about two kb long.

112. The T-cell of claim 110 wherein the antisense sequence binds to at least part of the HIV env gene.

113. The T-cell of claim 112 wherein the shRNA down-regulates the cell's HIV

CCR5 co-receptors.

114. The T-cell of claim 113 wherein the shRNA is embedded within another RNA sequence.

115. The T-cell of claim 107 wherein the heterologous fate-controlling sequence is selected from the group consisting of a cell-killing sequence and an anti-apoptosis sequence.

116. The T-cell of claim 115 wherein the cell-killing sequence comprises TK or dCK.

117. The T-cell of claim 115 wherein the cell-killing sequence comprises TMPK.

118. The T-cell of claim 115 wherein the anti-apoptosis sequence comprises a sequence that encodes a shRNA that targets the PDl gene.

119. The T-cell of claim 118 wherein the shRNA is embedded within another RNA sequence to enhance the processing of the shRNA.

120. The T-cell of claim 107 comprising two different heterologous anti-HIV sequences.

121. The T-cell of claim 120 wherein the anti-HIV sequences comprise a sequence that encodes an antisense RNA sequence that binds to at least part of the HIV genome and a sequence that encodes an miRNA that down-regulates at least one of the cell's

HIV co -receptors.

122. The T-cell of claim 121 wherein the antisense sequence binds to at least part of the HIV gag, pol, nef, or env gene and further includes a shRNA targeted to a highly conserved sequence of HIV.

123. The T-cell of claim 121 wherein the antisense sequence binds to at least part of the HIV env gene and the miRNA comprises an shRNA that down-regulates the cell's HIV CCR5 co-receptors.

124. The T-cell of claim 120 comprising two different heterologous fate-controlling sequences.

125. The T-cell of claim 124 wherein the fate-controlling sequences are selected from the group consisting of a cell-killing sequence, a cell selection sequence, and an anti-apoptosis sequence.

126. The T-cell of claim 125 wherein the cell-killing sequence comprises TMPK and the anti-apoptosis sequence comprises a sequence that encodes an shRNA that targets the PDl gene.

127. A T-cell comprising an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV env gene, a cell-killing sequence comprising TMPK, and an anti-apoptosis sequence that encodes an shRNA that targets the PDl gene.

128. The T-cell of claim 127 further comprising an anti-HIV sequence that encodes an shRNA that down-regulates the CCR5 HIV co-receptor on the surface of the cell.

129. The T-cell of any one of claims 107-128 wherein the anti-HIV sequences are anti-HIV- 1 sequences.

130. The T-cell of any one of claims 107-128 wherein the T-cell is allogeneic.

131. The T-cell of claim 130 wherein the anti-HIV sequences are anti-HIV-1 sequences.

132. A method of making an allogeneic or autologous human CD4⁺ T-cell that is resistant to HIV infection comprising the step of transducing an allogeneic or autologous human CD4⁺ T-cell with the lentiviral vector of any one of claims 1-31.

133. The method of claim 132 wherein the T-cell is an allogeneic T-cell.

134. The method of claim 133 wherein the allogeneic T-cell is obtained from human blood.

135. A method of making an allogeneic or autologous human CD4⁺ T-cell that is resistant to HIV-I infection comprising the step of transducing an allogeneic or autologous human CD4⁺ T-cell with the lentiviral vector claim 32.

136. The method of claim 135 wherein the T-cell is an allogeneic T-cell.

137. The method of claim 136 wherein the allogeneic T-cell is obtained from human blood.

138. The T-cell produced by the method of claim 132.

139. The T-cell produced by the method of claim 133.

140. The T-cell produced by the method of claim 134.

141. The T-cell produced by the method of claim 135.

142. The T-cell produced by the method of claim 136.

143. The T-cell produced by the method of claim 137.

144. An allogeneic or autologous human hematopoietic stem cell (HSC) comprising at least one heterologous anti-HIV sequence that inhibits HIV from infecting the cell or from replicating in the cell and further comprising at least one heterologous fate-controlling sequence.

145. The HSC of claim 144 wherein the anti-HIV sequence comprises a sequence that encodes an antisense RNA sequence that binds to at least part of the HIV genome or a sequence that encodes an miRNA that down-regulates at least one of the cell's HIV co -receptors.

146. The HSC of claim 145 wherein the antisense sequence binds to at least part of the HIV gag, pol, nef, or env gene.

147. The HSC of claim 145 wherein the antisense sequence binds to at least part of the HIV env gene.

148. The HSC of claim 147 wherein the co-receptor is CCR5.

149. The HSC of claim 144 wherein the heterologous fate-controlling sequence is selected from the group consisting of a cell selection sequence and a cell-killing sequence.

150. The HSC of claim 149 comprising two different heterologous anti-HIV sequences.

151. The HSC of claim 150 wherein the anti-HIV sequences comprise a sequence that encodes an antisense RNA sequence that binds to at least part of the HIV genome and a sequence that encodes an miRNA that down-regulates at least one of the cell's HIV co -receptors .

152. The HSC of claim 151 wherein the antisense sequence binds to at least part of the HIV env gene and the co-receptor is CCR5.

153. The HSC of claim 152 wherein the antisense sequence is about 500 bases to about 3000 bases long.

154. The HSC of claim 153 wherein the antisense sequence is about one kb to about two kb long.

155. The HSC of claim 152 wherein the miRNA comprises a shRNA.

156. The HSC of claim 149 wherein the cell selection sequence is selected from the group consisting of sequences encoding neomycin resistance protein, MDRl, and mutant DHFR.

157. The HSC of claim 149 wherein the cell selection sequence comprises MGMT.

158. The HSC of claim 149 wherein the cell-killing sequence comprises TK or dCK.

159. The HSC of claim 149 wherein the cell-killing sequence comprises TMPK.

160. The HSC of any one of claims 144-159 wherein the anti-HIV sequences are anti-HIV- 1 sequences.

161. The HSC of any one of claims 144-159 wherein the HSC is allogeneic.

162. The HSC of claim 161 wherein the anti-HIV sequences are anti-HIV- 1 sequences.

163. A method of making an allogeneic or autologous human HSC that is resistant to HIV infection comprising the step of transducing an allogeneic or autologous human HSC with the lentiviral vector of any one of claims 1-31.

164. The method of claim 163 wherein the cell is an allogeneic HSC.

165. The method of claim 164 wherein the allogeneic HSC is obtained from human bone marrow, human blood, human umbilical cord, or human umbilical cord blood.

166. A method of making an allogeneic or autologous human HSC that is resistant to HIV-I infection comprising the step of transducing an allogeneic or autologous human HSC with the lentiviral vector of claim 32.

167. The method of claim 166 wherein the cell is an allogeneic HSC.

168. The method of claim 167 wherein the allogeneic HSC is obtained from bone marrow, human blood, human umbilical cord, or human umbilical cord blood.

169. The HSC produced by the method of claim 163.

170. The HSC produced by the method of claim 164.

171. The HSC produced by the method of claim 165.

172. The HSC produced by the method of claim 166.

173. The HSC produced by the method of claim 167.

174. The HSC produced by the method of claim 168.

175. An allogeneic or autologous human MSC comprising at least one heterologous anti-HIV sequence that inhibits HIV from infecting the cell or from replicating in the cell and further comprising at least one heterologous fate-controlling sequence.

176. The MSC of claim 175 wherein the anti-HIV sequence comprises a sequence that encodes an antisense RNA sequence that binds to at least part of the HIV genome.

177. The MSC of claim 176 wherein the antisense sequence binds to at least part of the HIV env gene.

178. The MSC of claim 177 wherein the antisense sequence is about 500 bases to about 3000 kb long.

179. The MSC of claim 178 wherein the antisense sequence is about one kb to about two kb long.

180. The MSC of claim 175 wherein the fate-controlling sequence comprises a cell- killing sequence.

181. The MSC of claim 180 wherein the cell-killing sequence comprises TK or dCK.

182. The MSC of claim 180 wherein the cell-killing sequence comprises TMPK.

183. The MSC of claim 175 further comprising a sequence encoding a stem cell promotion factor.

184. The MSC of claim 183 wherein the stem cell promotion factor comprises a cytokine.

185. The MSC of claim 184 wherein the cytokine is selected from the group consisting of TPO, SCF, EPO, FLT-3L, IL-3, G-CSF, and GM-CSF.

186. The MSC of claim 184 wherein the cytokine comprises SCF, TPO, FLT-3L, or G-CSF.

187. The MSC of claim 183 wherein the stem cell promotion factor comprises H0X-B4.

188. The MSC of any one of claims 175-187 wherein the anti-HIV sequences are anti-HIV- 1 sequences.

189. The MSC of any one of claims 175-187 wherein the MSC is allogeneic.

190. The MSC of claim 189 wherein the anti-HIV sequences are anti-HIV- 1 sequences.

191. A method of making a genetically modified allogeneic or autologous human MSC comprising the step of transducing an allogeneic or autologous human MSC with a lentiviral vector comprising a heterologous anti-HIV sequence that inhibits HIV from infecting the cell or from replicating in the cell, a heterologous fate- controlling sequence, and a heterologous sequence encoding a stem cell promotion factor.

192. The method of claim 191 wherein the anti-HIV sequence comprises a sequence that encodes an antisense RNA sequence that binds to at least part of the HIV genome.

193. The method of claim 192 wherein the antisense sequence binds to at least part of the HIV env gene.

194. The method of claim 193 wherein the antisense sequence is about 500 bases to about 3000 kb long.

195. The method of claim 194 wherein the antisense sequence is about one kb to two kb long.

196. The method of claim 191 wherein the fate-controlling sequence comprises a cell-killing sequence.

197. The method of claim 196 wherein the cell-killing sequence comprises TK or dCK.

198. The method of claim 196 wherein the cell-killing sequence comprises TMPK.

199. The method of claim 191 wherein the stem cell promotion factor comprises a cytokine.

200. The method of claim 199 wherein the cytokine is selected from the group consisting of TPO, SCF, EPO, FLT-3L, IL-3, G-CSF, and GM-CSF.

201. The method of claim 199 wherein the cytokine comprises SCF, TPO, FLT-3L, or G-CSF.

202. The method of claim 191 wherein the stem cell promotion factor comprises H0X-B4.

203. The method of any one of claims 191-202 the anti-HIV sequences are anti- HIV-I sequences.

204. The method of any one of claims 191-202 wherein the MSC is an allogeneic MSC.

205. The method of claim 204 wherein the anti-HIV sequences are anti-HIV- 1 sequences.

206. The method of claim 205 wherein the allogeneic MSC is obtained from human bone marrow, human blood, human umbilical cord, or human umbilical cord blood.

207. The method of claim 206 wherein the MSC is a Human Umbilical Cord Perivascular Cell (HUCPVC).

208. The MSC produced by the method of claim 191.

209. The MSC produced by the method of claim 192.

210. The MSC produced by the method of claim 193.

211. The MSC produced by the method of claim 194.

212. The MSC produced by the method of claim 195.

213. The MSC produced by the method of claim 196.

214. The MSC produced by the method of claim 197.

215. The MSC produced by the method of claim 198.

216. The MSC produced by the method of claim 199.

217. The MSC produced by the method of claim 200.

218. The MSC produced by the method of claim 201.

219. The MSC produced by the method of claim 202.

220. The MSC produced by the method of claim 203.

221. The MSC produced by the method of claim 204.

222. The MSC produced by the method of claim 205.

223. The MSC produced by the method of claim 206.

224. The MSC produced by the method of claim 207.

225. A composition comprising an allogeneic or autologous human CD4⁺ T-cell, an allogeneic or autologous human HSC, and an allogeneic or autologous human MSC, wherein each cell comprises at least one heterologous anti-HIV sequence that inhibits

HIV from infecting the cells or from replicating in the cells and at least one heterologous fate-controlling sequence.

226. The composition of claim 225 wherein the MSC further comprises at least one heterologous sequence encoding a stem cell promotion factor.

227. The composition of claim 226 wherein the anti-HIV sequence comprises a sequence that encodes an antisense RNA sequence that binds to at least part of the HIV genome or a sequence that encodes an miRNA that down-regulates at least one of the cell's HIV co-receptors.

228. The composition of claim 227 wherein the antisense sequence binds to at least part of the HIV gag, pol, nef, or env gene.

229. The composition of claim 228 wherein the antisense sequence binds to at least part of the HIV env gene and the miRNA comprises an shRNA that down-regulates the cell's CCR5 HIV co-receptor.

230. The composition of claim 226 wherein the heterologous fate-controlling sequence is selected from the group consisting of a cell-killing sequence, a cell selection sequence, and an anti-apoptosis sequence.

231. The composition of claim 230 wherein the cell -killing sequence comprises TK or dCK.

232. The composition of claim 230 wherein the cell -killing sequence comprises TMPK.

233. The composition of claim 230 wherein the cell selection sequence is selected from the group consisting of sequences encoding neomycin resistance protein, MDRl, and DHFR.

234. The composition of claim 230 wherein the cell selection sequence comprises MGMT.

235. The composition of claim 230 wherein the anti-apoptosis sequence comprises a sequence that encodes an shRNA that targets the PDl gene.

236. The composition of claim 226 wherein the stem cell promotion factor comprises a ctokine.

237. The composition of claim 236 wherein the cytokine is selected from the group consisting of TPO, SCF, EPO, FLT-3L, IL-3, G-CSF, and GM-CSF.

238. The composition of claim 236 wherein the cytokine comprises SCF, TPO,

FLT-3L, or G-CSF.

239. The composition of claim 226 wherein the stem cell promotion factor comprises H0X-B4.

240. The composition of claim 226 wherein the T-cell and the HSC comprise two different anti-HIV sequences that inhibit HIV from infecting the cell or from replicating in the infected cell.

241. The composition of claim 240 wherein one anti-HIV sequence encodes an antisense RNA sequence that binds to at least part of the HIV genome and the other anti-HIV sequence encodes an miRNA that down-regulates at least one of the cell's HIV co -receptors .

242. The composition of claim 241 wherein the antisense sequence binds to at least part of the HIV gag, pol, nef, or env gene.

243. The composition of claim 242 wherein the antisense sequence binds to at least part of the HIV env gene and the miRNA comprises an shRNA that down-regulates the cell's CCR5 HIV co-receptor.

244. The composition of any one of claims 225-243 wherein the anti-HIV sequences are anti-HIV- 1 sequences.

245. The composition of any one of claims 225-243 wherein the cells are allogeneic cells.

246. The composition of claim 245 wherein the anti-HIV sequences are anti-HIV- 1 sequences.

247. A method of lowering the HIV viral load in a human comprising administering to the human an effective amount of the composition of any one of claims 225-243 wherein the cells of the composition engraft in the human, resulting in a decreased viral load in the human.

248. The method of claim 247 wherein the anti-HIV sequences are anti-HIV- 1 sequences.

249. The method of claim 247 wherein the cells are allogeneic cells.

250. The method of claim 249 wherein the anti-HIV sequences are anti-HIV-1 sequences.

251. A composition comprising an allogeneic or autologous human CD4⁺ T-cell and an allogeneic or autologous human HSC wherein each cell comprises at least one heterologous anti-HIV sequence that inhibits HIV from infecting the cells or from replicating in the cells and at least one heterologous fate-controlling sequence.

252. A composition comprising an allogeneic or autologous human CD4⁺ T-cell and an allogeneic or autologous human MSC, wherein each cell comprises at least one heterologous anti-HIV sequence that inhibits HIV from infecting the cells or from replicating in the cells and at least one heterologous fate-controlling sequence.

253. The composition of claim 252 wherein the MSC further comprises at least one heterologous sequence encoding a stem cell promotion factor.

254. A composition comprising an an allogeneic or autologous human HSC and an allogeneic or autologous human MSC wherein each cell comprises at least one heterologous anti-HIV sequence that inhibits HIV from infecting the cells or from replicating in the cells and at least one heterologous fate-controlling sequence.

255. The composition of claim 254 wherein the MSC further comprises at least one heterologous sequence encoding a stem cell promotion factor.

256. The composition of any one of claims 251 -255 wherein the anti-HIV sequence comprises a sequence that encodes an antisense RNA sequence that binds to at least part of the HIV genome or a sequence that encodes an miRNA that down-regulates at least one of the cell's HIV co-receptors.

257. The composition of claim 256 wherein the antisense sequence binds to at least part of the HIV gag, pol, nef, or env gene.

258. The composition of claim 257 wherein the antisense sequence binds to at least part of the HIV env gene and the miRNA comprises an shRNA that down-regulates the cell's CCR5 HIV co-receptor.

259. The composition of any one of claims 251 -255 wherein the heterologous fate- controlling sequence is selected from the group consisting of a cell-killing sequence, a cell selection sequence, and an anti-apoptosis sequence.

260. The composition of claim 259 wherein the cell -killing sequence comprises TK or dCK.

261. The composition of claim 259 wherein the cell -killing sequence comprises TMPK.

262. The composition of claim 259 wherein the cell selection sequence is selected from the group consisting of sequences encoding neomycin resistance protein, MDRl, and DHFR.

263. The composition of claim 259 wherein the cell selection sequence comprises MGMT.

264. The composition of claim 259 wherein the anti-apoptosis sequence comprises a sequence that encodes an shRNA that targets the PDl gene.

265. The composition of claim 253 or claim 255 wherein the stem cell promotion factor comprises a ctokine.

266. The composition of claim 265 wherein the cytokine is selected from the group consisting of TPO, SCF, EPO, FLT-3L, IL-3, G-CSF, and GM-CSF.

267. The composition of claim 265 wherein the cytokine comprises SCF, TPO, FLT-3L, or G-CSF.

268. The composition of claim 253 or 255 wherein the stem cell promotion factor comprises H0X-B4.

269. A lentiviral vector comprising an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV env gene, an anti-HIV sequence that encodes an miRNA embedded shRNA that down-regulates the CCR5 HIV co- receptors on the surface of a cell that can be infected by HIV, and a cell selection sequence comprising MGMT.

270. The lentiviral vector of claim 269 further comprising a cell-killing sequence comprising TMPK.

271. A lentiviral vector comprising an anti-HIV sequence that encodes an antisense

RNA sequence that binds to at least part of the HIV env gene, a cell-killing sequence comprising TMPK, and an anti-apoptosis sequence comprising a sequence that encodes an miRNA embedded shRNA that targets the PD-I gene.

272. The lentiviral vector of claim 271 further comprising an anti-HIV sequence that encodes a shRNA that down-regulates the CCR5 co-receptors on the surface of a cell that can be infected by HIV.

273. A lentiviral vector comprising an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV env gene, a cell-killing sequence comprising TMPK, and a sequence encoding the cytokines TPO, SCF, and FLT-3L.

274. A lentiviral vector comprising an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV env gene, a cell-killing sequence comprising TMPK, and a sequence encoding G-CSF.

275. A lentiviral vector comprising an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV env gene, a cell-killing sequence comprising TMPK, and a sequence encoding H0X-B4.

276. The lentiviral vector of any one of claims 269-275 wherein the anti-HIV sequences are anti-HIV- 1 sequences.

277. A plasmid comprising an antisense sequence that binds to at least part of the HIV env gene, anti-HIV sequence that encodes a shRNA that down-regulates the CCR5 HIV co-receptors on the surface of a cell that can be infected by HIV, and a cell selection sequence comprising MGMT.

278. The plasmid of claim 277 further comprising a cell-killing sequence comprising TMPK.

279. A plasmid comprising an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV env gene, a cell-killing sequence comprising TMPK, and an anti-apoptosis sequence comprising a sequence that encodes an shRNA that targets the PDl gene.

280. The plasmid of claim 279 further comprising an anti-HIV sequence that encodes an miRNA that down-regulates the CCR5 co-receptors on the surface of a cell that can be infected by HIV.

281. A plasmid comprising an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV env gene, a cell-killing sequence comprising TMPK, and a sequence encoding the cytokines TPO, SCF, and FLT-3L.

282. A plasmid comprising an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV env gene, a cell-killing sequence comprising TMPK, and a sequence encoding G-CSF.

283. A plasmid comprising an anti-HIV sequence that encodes an antisense RNA sequence that binds to at least part of the HIV env gene, a cell-killing sequence comprising TMPK, and a sequence encoding H0X-B4.

. The plasmid of any one of claims 277-283 wherein the anti-HIV sequences are anti-HIV-1 sequences.