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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Curr Opin Immunol. Author manuscript; available in PMC 2013 October 1.
Published in final edited form as:
PMCID: PMC3478429

Creating Genetic Resistance to HIV


HIV/AIDS remains a chronic and incurable disease, in spite of the notable successes of highly active antiretroviral therapy. Gene therapy offers the prospect of creating genetic resistance to HIV that supplants the need for antiviral drugs. In sight of this goal, a variety of anti-HIV genes have reached clinical testing, including gene-editing enzymes, protein-based inhibitors, and RNA-based therapeutics. Combinations of therapeutic genes against viral and host targets are designed to improve the overall antiviral potency and reduce the likelihood of viral resistance. In cell-based therapies, therapeutic genes are expressed in gene modified T lymphocytes or in hematopoietic stem cells that generate an HIV-resistant immune system. Such strategies must promote the selective proliferation of the transplanted cells and the prolonged expression of therapeutic genes. This review focuses on the current advances and limitations in genetic therapies against HIV, including the status of several recent and ongoing clinical studies.

Keywords: HIV, gene therapy, HSCs


The antiretroviral drug cocktail known as combination antiretroviral therapy (cART) potently inhibits HIV replication, reduces the rates of transmission, and enhances the quality and duration of life of patients. However, cART does not fully restore health and patients still experience chronic inflammation, immunosenescence, and increased risks of non-AIDS morbidity and mortality [1, 2]. It requires daily compliance and lifelong adherence, indefinitely extending the issues of side effects, cost, and drug resistance. Finally, cART does not eliminate latent reservoirs of virus in patients [3] and may fail to suppress completely viral replication despite drug intensification strategies [4, 5].

As an alternative to cART, gene therapy aims to confer “intracellular immunization” by genetically engineering cells for HIV resistance [6]. Such approaches typically utilize one or more therapeutic genes that inhibit steps within the viral infection and replication cycle including RNA-based therapeutics, gene editing enzymes, or protein inhibitors to target viral or host factors. Genetic resistance to HIV in protected cells may be accomplished by encoding therapeutic genes within an integrating vector, such as a retroviral, lentiviral, foamy viral, or transposon vector [7]. Alternatively, endogenous genes that are required for HIV infection and not normal cell function can be mutated or deleted using site-specific enzymes. Many anti-HIV gene therapy strategies rely on cell-based delivery, including the ex vivo transduction and subsequent transplantation of cells that are natural targets for HIV infection, including CD4+ T lymphocytes and macrophages. Similarly, the transplantation of genetically modified CD34+ hematopoietic stem cells (HSCs) offers further advantages, such as the ability for self-renewal and differentiation into all hematopoietic lineages, including CD4+ T cells, macrophages, and dendritic cells. Likewise, induced pluripotent stem cells (iPSCs) can be reprogrammed into HSCs and further engineered for HIV resistance [8, 9].

Gene therapy targets against HIV

HIV encodes nine viral genes (gag, pol, vif, vpr, tat, rev, vpu, env, and nef) that control all steps of the viral replicative cycle including entry into the targeted cells, reverse transcription, integration into the host genome, transcription of viral mRNA, assembly of viral proteins, and budding of the newly formed virions. Each of these genes and steps within the viral replication cycle serves as a potential target for novel gene therapeutics. Moreover, gene-based strategies offer the possibility of inhibiting viral genes at the level of DNA, RNA, or protein, thereby expanding the scope of druggable targets for HIV. Such strategies typically require the insertion of therapeutic genes into uninfected cells, though many antiviral genes also inhibit viral replication in infected cells. However, like cART, antiviral gene therapies are susceptible to mutational escape, as the virus can mutate its genome at positions targeted by the therapeutics.

Endogenous genes that are required for HIV infection and/or replication offer several advantages over viral targets. In contrast to viral targets, host genes are not as prone to escape from mutation or saturating levels of virus. Second, host factors may be genetically modified in cells prior to HIV infection, thereby “immunizing” them from the virus. Nevertheless, the virus may evolve to bypass the need for certain host factors, such as the CCR5 co-receptor, or it may produce viral proteins that directly combat host restriction factors (Vif and Vpu for APOBEC 3G and Tetherin).

Design principles for anti-HIV gene therapy

Theoretical models predict that therapeutic genes that inhibit HIV steps prior to integration (Class I) are more effective in promoting the survival and expansion of protected cells than agents that inhibit latter stages of the HIV replication cycle such as viral gene expression (Class II) or viral assembly and budding (Class III) [10, 11]. Importantly, these predictions are consistent with a recent in vivo study that concluded that the maC46 fusion inhibitor resulted in higher positive selection of transduced cells than an antisense transcript against the HIV-1 env mRNA or a short-hairpin RNA (shRNA) against the HIV tat/rev mRNA [12]. Similarly, another computational study investigated several key design principles for effective gene therapy strategies including the potency of viral inhibition in protected cells, the proliferation enhancement of protected cells, the potential value of splitting multiple therapies across cells, and the benefits of small but significant fitness costs associated with viral escape mutations [13].

Hope for a cure (Berlin patient)

In addition to the presence of the CD4 surface receptor on T lymphocytes, HIV requires either co-receptor chemokine receptor type 5 (CCR5) or chemokine receptor type 4 (CXCR4) for viral entry. Inhibition of either CCR5 or CXCR4 co-receptors by gene therapy has been extensively studied in laboratory and animal models of HIV infection, and several therapeutic applications for CCR5 knockdown have reached clinical trials [7]. In fact, CCR5 offers a promising target for anti-HIV therapeutics, as it is not essential for normal T cell function and homozygous carriers of a CCR5 partial gene deletion (CCR5Δ32) are naturally resistant to HIV infection. The concept of targeting CCR5 by gene therapy is bolstered by the famous “Berlin patient” study, in which an HIV patient with acute myeloid leukemia (AML) received CD34+ hematopoietic stem cells (HSCs) from a donor with a naturally occurring homozygous mutation in the CCR5 gene (CCR5Δ32), which led to the elimination of all detectable HIV from the patient after 4 years and an apparent functional “cure” of HIV infection [14, 15]. The transplanted CD34+ HSCs differentiated into all hematopoietic lineages, including HIV-resistant CD4+ lymphocytes and macrophages. Due to the preexisting AML, this patient required an allogeneic bone marrow transplant with complete bone marrow ablation and immune suppression, although these invasive procedures are not justifiable options for the treatment of HIV. Furthermore, due to the extremely low frequency of individuals who carry the CCR5Δ32 allele (5%–14% in individuals of European descent and much less common in individuals of African and Asian descent), finding an HLA-matched donor with HIV-resistant allele is an extraordinarily rare achievement [16]. It is likely that the graft versus host allogeneic effect was critical for elimination of detectible HIV in this patient, and this raises the question of whether gene modified autologous blood stem cells could be as successful. It might be necessary to engineer a selectable marker into such modified cells to ensure adequate expansion of protected progeny, as has been shown in a non-human primate model [17]. Thus, though the case of the “Berlin patient” is not directly applicable for other HIV patients, it strongly supports the notion for an HIV cure and underscores the potential of creating HIV resistance using many of the gene therapy strategies discussed forthwith.

Intracellular Immunization

Gene editing enzymes

The recent development of site-specific DNA editing proteins, including zinc-finger nucleases (ZFNs), has fueled the interest of permanently altering and inactivating a range of host or viral genes necessary for HIV infection and replication [1]. One of the earliest and most promising gene editing therapeutics is a ZFN from Sangamo BioSciences (Richmond, CA, USA) that disables the endogenous CCR5 gene by creating double-strand breaks and imperfect repair by nonhomologous end joining (NHEJ) at a location upstream of the natural CCR5Δ32 mutation [18]. This ZFN may be delivered ex vivo to human CD34+ HSCs or primary CD4+ T cells by adenoviral vectors [18], integrase-deficient lentiviral vectors [19], or nucleofection [20]. The adenoviral-delivered CCR5 ZFN in autologous CD4+ T cells, known as SB-728-T, is currently in clinical testing in two Phase 1 trials (NCT00842634 and NCT01044654) and two Phase 1/2 studies (NCT01543152 and NCT01252641). While all of these trials use the same SB-728-T therapy, they examine different patient cohorts, including viremic patients who have never received cART, viremic patients who have multidrug cART resistance, aviremic patients who remain on cART, and aviremic patients who have volunteered for scheduled treatment interruptions (STI). These multi-cohort studies aim to evaluate the efficacy and safety of SB-728-T, as well as to explore different conditions that might improve the engraftment and expansion of protected cells.

Although both CCR5 and CXCR4 ZFNs function independently, it might be necessary to eliminate both to immunize CD4+ T cells from HIV infection or to combine the CXCR4 ZFN therapy with the CCR5-specific fusion inhibitor maraviroc. Indeed, elimination of CXCR4 without the removal of CCR5 led to the eventual emergence of CCR5 (R5)-tropic virus in NSG mice transplanted with human CD4+ T cells [21]. Despite the risks associated with removing CXCR4 without also disrupting CCR5, adenoviral delivery of the CXCR4 ZFN provided superior protection from HIV infection over an integrated lentiviral vector expressing CXCR4 shRNAs in NSG mice transplanted with human CD4+ T cells [22].

While ZFN approaches have aimed at genetically modifying the host CCR5 and CXCR4 genes to prevent HIV infection, other gene editing approaches target the viral gene locus in HIV-infected cells. These include an engineered HIV-specific homing endonuclease [23] and an LTR-specific recombinase (Tre-recombinase) that recognizes asymmetric sequences within the 5’ and 3’ LTRs flanking the viral genes [24, 25]. Although such potential therapeutics exist in earlier development stages than their ZFN counterparts, they offer the ability to eliminate HIV from infected cells, including latently infected cells which are currently untreatable by any clinical therapy.

Protein-based inhibitors

Another strategy to block HIV entry utilizes a fusion inhibitor known as C46, a 46-amino acid cell membrane-anchored peptide derived from the second heptad repeat of the HIV envelope glycoprotein gp41 [26]. In a Phase I clinical trial of ten HIV-infected patients, expression of C46 from a gammaretroviral vector (M87o) in autologous CD4+ T cells persisted for up to a year post-transplantation with no observed adverse events [27]. A Phase I-II clinical study at the University Medical Center Hamburg-Eppendorf continues to evaluate the M87o vector in autologous CD34+ HSCs for patients with high-risk AIDS-related lymphoma (NCT00858793). Preclinical studies support the efficacy of the C46 peptide, as its expression from a lentiviral vector protected infection in HSCs in a macaque model of HIV [28] and in primary CD4+ T cells in a humanized mouse model [12]. Furthermore, the C46 peptide can also be expressed as a secreted antiviral entry inhibitor (SAVE), which provides a bystander effect for neighboring unmodified cells [29]. However, C46 is subject to resistance induction by selection of envelope mutations [30], and it will likely be necessary to use C46 as part of combinatorial gene therapy strategies.

In addition to the surface receptors required for viral entry, knockdown screens have identified hundreds of unique host genes that are necessary for HIV infection and replication and may offer targets for novel antiretroviral therapies [31]. One such factor required for HIV integration is the lens epithelium-derived growth factor (LEDGF/p75), and overexpression of the C-terminal portion that contains the HIV-1 integrase (IN)-binding domain (IDB) can inhibit viral replication in both human CD4+ T cells and in the humanized NSG mouse model [32

Alternatively, many types of nonhuman cells, such as murine and simian, are naturally resistant to HIV infection due to species-specific “restriction factors” that could be therapeutically exploited in gene therapy applications, such as APOBEC3G, tripartite motif 5α(TRIM5α), and BST-2/tetherin (reviewed in [33]). Among such restriction factors, engineered versions of APOBEC3G [3436] and TRIM5 α[37, 38] inhibit HIV infection and replication in preclinical gene therapy applications.

RNA-based therapeutics

RNA-based therapeutics offer several advantages as anti-HIV genes including high levels of expression, low immunogenicity, and design flexibility against a range of viral and cellular targets. Hence, several RNA-based strategies have reached early stages of clinical testing. RNA-based drugs may be classified by the mechanism of activity such as catalytically active RNA molecules (ribozymes), transcripts that inhibit RNA translation by Watson-Crick base-pairing (antisense), RNAs that bind to proteins and other molecular ligands (decoys/aptamers), and small interfering RNAs (siRNAs) that induce RNA interference (RNAi) [39].


Ribozymes are catalytically active RNAs that can be engineered to induce site-specific cleavage of target RNA molecules in cis (the same nucleic acid strand) or in trans (a noncovalently linked nucleic acid). The first clinical trials using ribozymes consisted of anti-HIV hairpin or hammerhead ribozymes encoded in the attenuated Moloney murine leukemia virus (MMLV) gammaretroviral vector that targeted well-conserved positions in HIV mRNA transcripts [40, 41]. The primary objectives of these trials were to assess the safety of the ribozyme and to determine whether the ribozyme imparted a survival advantaged for the protected cells. Two Phase I clinical trials have examined different anti- HIV ribozymes in autologous [40, 42] and syngeneic CD4+ T lymphocytes [41, 43], while a third anti- HIV ribozyme in autologous CD34+ hematopoietic progenitor cells (HPCs) has reached Phase II trials (NCT00074997 and NCT01177059) [44, 45]. While these trials demonstrated the safety and feasibility of gene-derived ribozymes, none demonstrated a clear survival advantage for the protected cells vs. the empty vector (control) transduced cells.


RNA antisense transcripts recognize and bind their cognate mRNA targets by Watson-Crick base pairing to inhibit the function of the sense viral mRNAs. By generating long dsRNA complexes with the target (sense) mRNA, antisense transcripts block the processing of viral mRNA transcripts, induce adenosine to inosine RNA editing by ADAR (adenosine deaminase that acts on RNA), and trigger the degradation of hyper-edited mRNA transcripts by inosine-specific nucleases [46]. VIRxSYS Corporation has developed a gene therapy approach against HIV-1 using a “conditionally replicating” lentiviral vector (VRX496) that encodes a 937 bp antisense sequence against the HIV env region of viral transcripts. While VRX496 does not encode any viral genes, it is engineered with full-length 5’ and 3’ long terminal repeats (LTR) and other essential structural nucleic acid elements that enable its replication only in cells also infected with wildtype HIV [47]. While the “conditionally replicating” feature of this gene therapy strategy is designed to provide additional therapeutic protection, highly efficient inhibition of HIV by the therapeutic vector may severely limit the mobilization and persistence of the vector [48, 49].

In a Phase I clinical trial for five HIV patients having resistance to multiple CART regimens, autologous CD4+ T cells were harvested from the patients, transduced ex vivo with the VRX496 antisense vector, and transplanted back into the patients. In this trial the T-cell-based infusion of VRX496, referred to as Lexgenleucel-T, was well tolerated for all patients with no detectable insertional mutagenesis, clonal outgrowth, or immunogenicity from the lentiviral vector [47]. The integrated VRX496 DNA was detected for at least one year in PBMCs from two subjects and VRX496 genomic RNA mobilization in plasma was observed for up to two months. Two follow-up Phase II trials (NCT00622232 and NCT00131560) in cART-failed subjects compared single or multiple doses of Lexgenleucel-T in modified CD4+ T cells, though multiple infusions did not confer significant immunological benefits.

RNA aptamer/decoy

RNA aptamers are single stranded molecules that bind target ligands with high affinity and specificity due to their stable three-dimensional structure. Aptamers can be generated and identified de novo through a selection process known as systematic evolution of ligands by exponential enrichment (SELEX) for a specific function, such as affinity for a particular protein ligand. Alternatively, RNA aptamers known as RNA decoys are mimics of natural RNA molecules that competitively target the cognate RNA-binding protein or ligand. Two RNA decoys that mimic either the HIV-1 Rev response element (RRE) or the transactivating region (TAR) have reached clinical trials as anti-HIV therapeutics. In the earliest anti-HIV RNA decoy trial, conducted by researchers at Children’s Hospital Los Angeles, autologous CD34+ HPCs were transduced ex vivo with a retroviral vector expressing the 41-nt RRE decoy, which is designed to bind and inhibit the HIV Rev protein [50]. Though no adverse events among the four HIV infected subjects were associated with the gene therapy, the low frequency of retroviral gene transfer and engraftment precluded conclusions about the anti-viral potency of the RNA decoy. A different clinical study from the City of Hope and Benitec, Inc used a nucleolar-localizing anti-HIV TAR decoy as one component of an all RNA-based gene therapy that also included a shRNA and a ribozyme [51]. All components of the anti-HIV therapy (pHIV7-shI-TAR-CCR5RZ) were expressed from a lentiviral vector in autologous CD34+ HPCs and transplanted in patients with AIDS-related non-Hodgkins lymphoma.


The cellular process of RNA interference (RNAi) refers to the conserved sequence-specific degradation of messenger RNA (mRNA) mediated by small double-stranded RNAs. Due to the high sequence-specificity of RNAi, synthetic small interfering (siRNAs) or their precursor short hairpin RNAs (shRNAs) can be designed to specifically target any complementary sequence within a particular gene. RNAi knockdown of a targeted mRNA, defined as post transcriptional gene silencing (PTGS), can be induced by delivering siRNA molecules directly to the cells as ~19–23 bp dsRNAs or by the de novo transcription shRNA precursors and their subsequent processing into siRNAs. For prolonged intracellular immunity, constitutive expression of anti-HIV shRNAs from integrated lentiviral vectors can induce long-term gene silencing for the duration of the shRNA transcription and biogenesis.

SiRNA/shRNAs against viral target sites may become ineffective with the emergence of nucleotide substitutions or deletions within the viral genome at the target site [52, 53] or mutations of the viral genome at sites outside of the target site that confer some fitness advantage for the virus under the selective pressure of RNAi [54, 55]. To minimize potential mutational escape from any specific siRNA, multiple shRNAs may be expressed by different RNA Pol III promoters or as a multicistronic transcript from the same promoter [5660].

Combinations of anti-HIV genes

Combinations of multiple anti-HIV genes in a single therapeutic vector or delivery system may increase the overall antiviral potency, reduce the required dosage level for each component, and decrease the potential for mutational escape. In contrast to combining multiple anti-HIV shRNAs into a single therapeutic, as discussed in the previous section, strategies that incorporate multiple types of therapeutics (ribozymes, decoys, etc.) minimize the competition and toxicity concerns associated with multiple shRNAs. Moreover, these strategies aim to inhibit HIV replication at multiple steps and typically include at least one Class I inhibitor, which inhibits early steps in the viral cycle prior to integration [7]. One design for a combinatorial lentiviral vector aimed to inhibit each of the three steps in the HIV replication cycle by expressing a CCR5 shRNA (pre-entry), a human/rhesus TRIM5α chimera (post-entry/pre- integration), and a TAR decoy (post-integration) [61]. This triple-agent vector was transduced into human CD34+ HSC transplanted NRG mice, leading to gene-modified CD4+ T cells and macrophages that were resistant to infection with either R5-tropic or CXCR4 (R4)-tropic HIV [62].

A different combinatorial strategy used a foamy virus vector to express the C46 envelope fusion inhibitor and two anti-HIV shRNAs against tat and rev viral genes and the endogenous ccr5 mRNA [63]. Additionally, the vector expresses the MGMTP140K gene from a separate promoter to select for gene modified cells in vivo. The vector was tested in human CD34+ HSCs transplanted in NSG mice and can also be used to inhibit simian immunodeficiency virus/HIV-1 (SHIV) chimera that is useful in monkey AIDS models.

To date, the only clinical trial to use a combinatorial gene therapy strategy against HIV was performed as a human pilot feasibility study from the City of Hope (Duarte California, USA) and Benitec (Melbourne, Australia). This study, which consisted of four patients with AIDS-related non-Hodgkin’s lymphoma (NHL), examined the safety and tolerability of autologous CD34+ HSCs modified with a replication incompetent lentiviral vector that encoded three anti-HIV small RNAs (pHIV7-shI-TAR-CCR5RZ) [51]. The cells were transduced ex vivo and then transplanted to express an shRNA against the overlapping reading frames of viral genes tat and rev, a nucleolar-localizing mimic of the viral RNA hairpin TAR that serves as a decoy that binds and sequesters the Tat protein, and a ribozyme targeting the mRNA of the endogenous CCR5 co-receptor. For all patients, the therapy was well tolerated and the siRNA was detected by quantitative PCR (qPCR) in primary blood mononuclear cells (PBMCs) and/or primary blood granulocytic cells (PBGCs) for at least 6 months post-treatment and beyond 36 months for one patient. Furthermore, levels of gene marking increased in two patients after increases in viremia, suggesting that the protected cells offered a selective advantage over unprotected cells in the presence of HIV [64].


With more than ten anti-HIV gene therapeutics having reached clinical trials and dozens more in preclinical testing, the future remains promising for HIV-resistant gene therapy. However, many issues remain unresolved for the development of efficient and robust gene therapy that provides comparable in vivo viral suppression to cART. Similar to the cocktail strategies of cART, many gene therapy applications for HIV use combinations of antiviral genes, including the usage of one or more Class I inhibitors. Such approaches aim to minimize the likelihood of mutational escape and enhance the proliferative capacity of the HIV-resistant engineered cells. However, careful design must ensure that the expression of antiviral genes does not exhibit cellular toxicity or confer any growth disadvantage to the modified cells, since the prolonged benefits of gene therapy require the engineered cells to replace the existing unmodified cells after transplantation. Myeoablative conditioning increases the engraftment capacity of transplanted HSCs and alternative non-myeoablative conditioning regimens are currently under exploration in various animal models [1]. Brief analytical treatment interruptions of cART may enhance in vivo selection of the protected cells and are useful in analyzing the antiviral efficacy of the gene therapy. Animal models are also proving useful to explore additional strategies for in vivo selection, such as the inclusion of chemically inducible survival factors [65, 66] or the co-expression of a chemotherapeutic resistance gene under drug selection [17, 63].

While the Berlin patient offers hope that HIV eradication may be achievable by gene therapy, this occurred in an allogeneic setting, and additional hurdles remain in adapting gene therapy for the general population of HIV patients using autologous cells. In fact, it remains to be determined whether, in the absence of an allogeneic effect, the HIV reservoir can be eliminated. Besides the technical issues surrounding the efficacy and duration of such cellular therapy, the significant safety and cost of such procedures must be less than those associated with a lifetime of cART. Moreover, although initial clinical studies support the safety of anti-HIV gene therapy, the general risks associated with cell transplantation and conditioning must improve for it to become a practical and feasible therapy for most HIV patients. Thus, current efforts in preclinical and clinical studies must continue to improve the efficacy, safety, and affordability of gene therapy schemes to realize the goal of creating genetic resistance for HIV.

Table 1
Anti-HIV gene therapeutics in clinical trials
Table 2
Anti-HIV gene therapeutics in pre-clinical animal studies


  • Gene therapy aims to create HIV-resistant cells to replace the need for cART.
  • Anti-HIV genes include protein or RNA-based agents and gene-editing enzymes.
  • Combinations of anti-HIV genes reduce the likelihood of viral resistance.
  • Current therapies require autologous transplantation of gene-modified cells.
  • Stem cell-based approaches aim to establish an HIV-resistant immune system.


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References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

* of special interest

** of outstanding interest

1. Kiem HP, Jerome KR, Deeks SG, McCune JM. Hematopoietic-stem-cell-based gene therapy for HIV disease. Cell Stem Cell. 2012;10:137–147. [PMC free article] [PubMed]
2. Deeks SG, Autran B, Berkhout B, Benkirane M, Cairns S, Chomont N, Chun TW, Churchill M, Mascio MD, Katlama C, et al. Towards an HIV cure: a global scientific strategy. Nat Rev Immunol. 2012 [PMC free article] [PubMed]
3. Siliciano JD, Kajdas J, Finzi D, Quinn TC, Chadwick K, Margolick JB, Kovacs C, Gange SJ, Siliciano RF. Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells. Nat Med. 2003;9:727–728. [PubMed]
4. Buzon MJ, Massanella M, Llibre JM, Esteve A, Dahl V, Puertas MC, Gatell JM, Domingo P, Paredes R, Sharkey M, et al. HIV-1 replication and immune dynamics are affected by raltegravir intensification of HAART-suppressed subjects. Nat Med. 2010;16:460–465. [PubMed]
5. Yukl SA, Shergill AK, McQuaid K, Gianella S, Lampiris H, Hare CB, Pandori M, Sinclair E, Gunthard HF, Fischer M, et al. Effect of raltegravir-containing intensification on HIV burden and T-cell activation in multiple gut sites of HIV-positive adults on suppressive antiretroviral therapy. AIDS. 2010;24:2451–2460. [PMC free article] [PubMed]
6. Baltimore D. Gene therapy. Intracellular immunization. Nature. 1988;335:395–396. [PubMed]
7. Scherer LJ, Rossi JJ. Ex vivo gene therapy for HIV-1 treatment. Hum Mol Genet. 2011;20:R100–R107. [PMC free article] [PubMed]
8. Kambal A, Mitchell G, Cary W, Gruenloh W, Jung Y, Kalomoiris S, Nacey C, McGee J, Lindsey M, Fury B, et al. Generation of HIV-1 resistant and functional macrophages from hematopoietic stem cell-derived induced pluripotent stem cells. Mol Ther. 2011;19:584–593. [PubMed]
9. Kamata M, Liu S, Liang M, Nagaoka Y, Chen IS. Generation of human induced pluripotent stem cells bearing an anti-HIV transgene by a lentiviral vector carrying an internal murine leukemia virus promoter. Hum Gene Ther. 2010;21:1555–1567. [PMC free article] [PubMed]
10. von Laer D, Hasselmann S, Hasselmann K. Impact of gene-modified T cells on HIV infection dynamics. J Theor Biol. 2006;238:60–77. [PubMed]
11. Applegate TL, Birkett DJ, McIntyre GJ, Jaramillo AB, Symonds G, Murray JM. In silico modeling indicates the development of HIV-1 resistance to multiple shRNA gene therapy differs to standard antiretroviral therapy. Retrovirology. 2010;7:83. [PMC free article] [PubMed]
12. Kimpel J, Braun SE, Qiu G, Wong FE, Conolle M, Schmitz JE, Brendel C, Humeau LM, Dropulic B, Rossi JJ, et al. Survival of the fittest: positive selection of CD4+ T cells expressing a membrane-bound fusion inhibitor following HIV-1 infection. PLoS ONE. 2010;5:e12357. [PMC free article] [PubMed]
13. Aviran S, Shah PS, Schaffer DV, Arkin AP. Computational models of HIV-1 resistance to gene therapy elucidate therapy design principles. PLoS Comput Biol. 2010;6 [PMC free article] [PubMed]
14. Allers K, Hutter G, Hofmann J, Loddenkemper C, Rieger K, Thiel E, Schneider T. Evidence for the cure of HIV infection by CCR5Delta32/Delta32 stem cell transplantation. Blood. 2011;117:2791–2799. [PubMed] ** The first and only evidence of a complete “cure” of HIV infection. The HIV patient with chronic myeloid leukemia remains free of any detectable HIV RNA and DNA 45 months after receiving a bone marrow transplant from a CCR5Δ32 donor, despite discontinuation of antiretroviral therapy.
15. Hutter G, Nowak D, Mossner M, Ganepola S, Mussig A, Allers K, Schneider T, Hofmann J, Kucherer C, Blau O, et al. Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. N Engl J Med. 2009;360:692–698. [PubMed]
16. Hutter G, Thiel E. Allogeneic transplantation of CCR5-deficient progenitor cells in a patient with HIV infection: an update after 3 years and the search for patient no. 2. Aids. 2011;25:273–274. [PubMed]
17. Beard BC, Trobridge GD, Ironside C, McCune JS, Adair JE, Kiem HP. Efficient and stable MGMT-mediated selection of long-term repopulating stem cells in nonhuman primates. J Clin Invest. 2010;120:2345–2354. [PMC free article] [PubMed]
18. Perez EE, Wang J, Miller JC, Jouvenot Y, Kim KA, Liu O, Wang N, Lee G, Bartsevich VV, Lee YL, et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol. 2008;26:808–816. [PMC free article] [PubMed]
19. Lombardo A, Genovese P, Beausejour CM, Colleoni S, Lee YL, Kim KA, Ando D, Urnov FD, Galli C, Gregory PD, et al. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat Biotechnol. 2007;25:1298–1306. [PubMed]
20. Holt N, Wang J, Kim K, Friedman G, Wang X, Taupin V, Crooks GM, Kohn DB, Gregory PD, Holmes MC, et al. Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo. Nat Biotechnol. 2010;28:839–847. [PMC free article] [PubMed]
21. Wilen CB, Wang J, Tilton JC, Miller JC, Kim KA, Rebar EJ, Sherrill-Mix SA, Patro SC, Secreto AJ, Jordan AP, et al. Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases. PLoS Pathog. 2011;7:e1002020. [PubMed] * This was the first study to use a cxcr4 zinc finger nuclease to create HIV-resistant CD4+ T cells. While the loss of CXCR4 protected CD4+ T cells in a humanized mouse model, R5-tropic HIV eventually emerged. The authors suggest that gene disruption of both cxcr4 and ccr5 may be necessary to prevent HIV resistance.
22. Yuan J, Wang J, Crain K, Fearns C, Kim KA, Hua KL, Gregory PD, Holmes MC, Torbett BE. Zinc-finger nuclease editing of human cxcr4 promotes HIV-1 CD4(+) T cell resistance and enrichment. Mol Ther. 2012;20:849–859. [PubMed] * In this study, CD4+ T cells were transduced with an adenoviral vector encoding a cxcr4 zinc finger nuclease and transplanted into the NSG mice. The ZFN-modified cells were resistant to an X4-tropic HIV strain and displayed a selective advantage in vivo.
23. Aubert M, Ryu BY, Banks L, Rawlings DJ, Scharenberg AM, Jerome KR. Successful targeting and disruption of an integrated reporter lentivirus using the engineered homing endonuclease Y2 I-AniI. PLoS ONE. 2011;6:e16825. [PMC free article] [PubMed]
24. Mariyanna L, Priyadarshini P, Hofmann-Sieber H, Krepstakies M, Walz N, Grundhoff A, Buchholz F, Hildt E, Hauber J. Excision of HIV-1 proviral DNA by recombinant cell permeable tre-recombinase. PLoS ONE. 2012;7:e31576. [PMC free article] [PubMed]
25. Sarkar I, Hauber I, Hauber J, Buchholz F. HIV-1 proviral DNA excision using an evolved recombinase. Science. 2007;316:1912–1915. [PubMed]
26. Egelhofer M, Brandenburg G, Martinius H, Schult-Dietrich P, Melikyan G, Kunert R, Baum C, Choi I, Alexandrov A, von Laer D. Inhibition of human immunodeficiency virus type 1 entry in cells expressing gp41-derived peptides. J Virol. 2004;78:568–575. [PMC free article] [PubMed]
27. van Lunzen J, Glaunsinger T, Stahmer I, von Baehr V, Baum C, Schilz A, Kuehlcke K, Naundorf S, Martinius H, Hermann F, et al. Transfer of autologous gene-modified T cells in HIV-infected patients with advanced immunodeficiency and drug-resistant virus. Mol Ther. 2007;15:1024–1033. [PubMed]
28. Trobridge GD, Wu RA, Beard BC, Chiu SY, Munoz NM, von Laer D, Rossi JJ, Kiem HP. Protection of stem cell-derived lymphocytes in a primate AIDS gene therapy model after in vivo selection. PLoS ONE. 2009;4:e7693. [PMC free article] [PubMed]
29. Egerer L, Volk A, Kahle J, Kimpel J, Brauer F, Hermann FG, von Laer D. Secreted antiviral entry inhibitory (SAVE) peptides for gene therapy of HIV infection. Mol Ther. 2011;19:1236–1244. [PubMed] * This study engineered a secreted HIV-fusion inhibitor by expressing two C46 peptides as concatemers connected by a furin cleavage motif. When present in low nanomolar concentrations, the secreted antiviral peptides inhibited HIV infection in Jurkat T cells and human primary T cells.
30. Hermann FG, Egerer L, Brauer F, Gerum C, Schwalbe H, Dietrich U, von Laer D. Mutations in gp120 contribute to the resistance of human immunodeficiency virus type 1 to membrane-anchored C-peptide maC46. J Virol. 2009;83:4844–4853. [PMC free article] [PubMed]
31. Goff SP. Knockdown screens to knockout HIV-1. Cell. 2008;135:417–420. [PMC free article] [PubMed]
32. Vets S, Kimpel J, Volk A, De Rijck J, Schrijvers R, Verbinnen B, Maes W, Von Laer D, Debyser Z, Gijsbers R. Lens Epithelium-derived Growth Factor/p75 Qualifies as a Target for HIV Gene Therapy in the NSG Mouse Model. Mol Ther. 2012;20:908–917. [PubMed] * In this study, human CD4+ T cells were transduced ex vivo with a lentiviral vector encoding the LEDGF325–530 peptide and engrafted into the NSG mouse. Expression of the antiviral peptide in CD4+ T cells reduced HIV replication and supported the survival of protected cells.
33. Malim MH, Bieniasz PD. HIV Restriction Factors and Mechanisms of Evasion. Cold Spring Harb Perspect Med. 2012;2:a006940. [PMC free article] [PubMed]
34. Wang Y, Shao Q, Yu X, Kong W, Hildreth JE, Liu B. N-terminal hemagglutinin tag renders lysine-deficient APOBEC3G resistant to HIV-1 Vif-induced degradation by reduced polyubiquitination. J Virol. 2011;85:4510–4519. [PMC free article] [PubMed]
35. Li L, Liang D, Li JY, Zhao RY. APOBEC3G-UBA2 fusion as a potential strategy for stable expression of APOBEC3G and inhibition of HIV-1 replication. Retrovirology. 2008;5:72. [PMC free article] [PubMed]
36. Ao Z, Wang X, Bello A, Jayappa KD, Yu Z, Fowke K, He X, Chen X, Li J, Kobinger G, et al. Characterization of anti-HIV activity mediated by R88-APOBEC3G mutant fusion proteins in CD4+ T cells, peripheral blood mononuclear cells, and macrophages. Hum Gene Ther. 2011;22:1225–1237. [PubMed] * This study tested the anti-HIV potency of APOBEC3G mutants fused with the virus-targeting polypeptide R88. The most potent fusion protein inhibited virus in PBMCs and macrophages when expressed from a recombinant adeno associated virus (AAV) vector.
37. Anderson J, Akkina R. Human immunodeficiency virus type 1 restriction by human-rhesus chimeric tripartite motif 5alpha (TRIM 5alpha) in CD34(+) cell-derived macrophages in vitro and in T cells in vivo in severe combined immunodeficient (SCID-hu) mice transplanted with human fetal tissue. Hum Gene Ther. 2008;19:217–228. [PubMed]
38. Neagu MR, Ziegler P, Pertel T, Strambio-De-Castillia C, Grutter C, Martinetti G, Mazzucchelli L, Grutter M, Manz MG, Luban J. Potent inhibition of HIV-1 by TRIM5-cyclophilin fusion proteins engineered from human components. J Clin Invest. 2009;119:3035–3047. [PMC free article] [PubMed]
39. Burnett JC, Rossi JJ. RNA-Based Therapeutics: Current Progress and Future Prospects. Chemistry & Biology. 2012;19:60–71. [PMC free article] [PubMed]
40. Wong-Staal F, Poeschla EM, Looney DJ. A controlled, Phase 1 clinical trial to evaluate the safety and effects in HIV-1 infected humans of autologous lymphocytes transduced with a ribozyme that cleaves HIV-1 RNA. Hum Gene Ther. 1998;9:2407–2425. [PubMed]
41. Cooper D, Penny R, Symonds G, Carr A, Gerlach W, Sun LQ, Ely J. A marker study of therapeutically transduced CD4+ peripheral blood lymphocytes in HIV discordant identical twins. Hum Gene Ther. 1999;10:1401–1421. [PubMed]
42. Feng Y, Leavitt M, Tritz R, Duarte E, Kang D, Mamounas M, Gilles P, Wong-Staal F, Kennedy S, Merson J, et al. Inhibition of CCR5-dependent HIV-1 infection by hairpin ribozyme gene therapy against CC-chemokine receptor 5. Virology. 2000;276:271–278. [PubMed]
43. Macpherson JL, Boyd MP, Arndt AJ, Todd AV, Fanning GC, Ely JA, Elliott F, Knop A, Raponi M, Murray J, et al. Long-term survival and concomitant gene expression of ribozyme-transduced CD4+ T-lymphocytes in HIV-infected patients. J Gene Med. 2005;7:552–564. [PubMed]
44. Amado RG, Mitsuyasu RT, Rosenblatt JD, Ngok FK, Bakker A, Cole S, Chorn N, Lin LS, Bristol G, Boyd MP, et al. 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. [PubMed]
45. Mitsuyasu RT, Merigan TC, Carr A, Zack JA, Winters MA, Workman C, Bloch M, Lalezari J, Becker S, Thornton L, et al. Phase 2 gene therapy trial of an anti-HIV ribozyme in autologous CD34+ cells. Nat Med. 2009;15:285–292. [PMC free article] [PubMed]
46. Faghihi MA, Wahlestedt C. Regulatory roles of natural antisense transcripts. Nat Rev Mol Cell Biol. 2009;10:637–643. [PMC free article] [PubMed]
47. Levine BL, Humeau LM, Boyer J, MacGregor RR, Rebello T, Lu X, Binder GK, Slepushkin V, Lemiale F, Mascola JR, et al. Gene transfer in humans using a conditionally replicating lentiviral vector. Proc Natl Acad Sci U S A. 2006;103:17372–17377. [PubMed]
48. Metzger VT, Lloyd-Smith JO, Weinberger LS. Autonomous targeting of infectious superspreaders using engineered transmissible therapies. PLoS Comput Biol. 2011;7:e1002015. [PubMed] * This computational study predicts that the ideal conditionally replicating virus must have a significantly higher expression rate (up to 20-fold) over wildtype virus in a co-infected cell, but does not require strong inhibition of HIV in order to maintain mobilization of the therapeutic virus.
49. Weinberger LS, Schaffer DV, Arkin AP. Theoretical design of a gene therapy to prevent AIDS but not human immunodeficiency virus type 1 infection. J Virol. 2003;77:10028–10036. [PMC free article] [PubMed]
50. Kohn DB, Bauer G, Rice CR, Rothschild JC, Carbonaro DA, Valdez P, Hao Q, Zhou C, Bahner I, Kearns K, et al. A clinical trial of retroviral-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. [PubMed]
51. DiGiusto DL, Krishnan A, Li L, Li H, Li S, Rao A, Mi S, Yam P, Stinson S, Kalos M, et al. RNA-based gene therapy for HIV with lentiviral vector-modified CD34(+) cells in patients undergoing transplantation for AIDS-related lymphoma. Sci Transl Med. 2010;2:36ra43. [PMC free article] [PubMed] ** This clinical study examined the safety and feasibility of expressing three RNA-based anti-HIV genes in autologous CD34+ HSCs. Four patients with AIDS-related lymphoma received transplantations of both HIV-resistant and unmodified HSCs. Two of these patients exhibited prolonged expression of the antiviral genes up to 18 and 24 months, respectively.
52. Das AT, Brummelkamp TR, Westerhout EM, Vink M, Madiredjo M, Bernards R, Berkhout B. Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition. J Virol. 2004;78:2601–2605. [PMC free article] [PubMed]
53. Sabariegos R, Gimenez-Barcons M, Tapia N, Clotet B, Martinez MA. Sequence homology required by human immunodeficiency virus type 1 to escape from short interfering RNAs. J Virol. 2006;80:571–577. [PMC free article] [PubMed]
54. Shah PS, Pham NP, Schaffer DV. HIV develops indirect cross-resistance to combinatorial RNAi targeting two distinct and spatially distant sites. Mol Ther. 2012;20:840–848. [PubMed] * This study indicates that HIV can develop resistance to specific antiviral shRNAs with the emergence of mutations in the untranscribed promoter region, suggesting that the virus can indirectly escape the general RNAi mechanism by overwhelming it with increased numbers of HIV transcripts.
55. Leonard JN, Shah PS, Burnett JC, Schaffer DV. HIV evades RNA interference directed at TAR by an indirect compensatory mechanism. Cell Host Microbe. 2008;4:484–494. [PMC free article] [PubMed]
56. Saayman S, Arbuthnot P, Weinberg MS. Deriving four functional anti-HIV siRNAs from a single Pol III-generated transcript comprising two adjacent long hairpin RNA precursors. Nucleic Acids Res. 2010;38:6652–6663. [PMC free article] [PubMed]
57. ter Brake O, t Hooft K, Liu YP, Centlivre M, von Eije KJ, Berkhout B. Lentiviral vector design for multiple shRNA expression and durable HIV-1 inhibition. Mol Ther. 2008;16:557–564. [PubMed]
58. Zhang J, Rossi JJ. Strategies in designing multigene expression units to downregulate HIV-1. Methods Mol Biol. 2010;623:123–136. [PubMed]
59. Schopman NC, ter Brake O, Berkhout B. Anticipating and blocking HIV-1 escape by second generation antiviral shRNAs. Retrovirology. 2010;7:52. [PMC free article] [PubMed]
60. Chung J, Zhang J, Li H, Ouellet D, Digiusto DL, Rossi JJ. Endogenous MCM7 miRNA Cluster as a Novel Platform to Multiplex Small Interfering and Nucleolar RNAs for Combinational HIV-1 Gene Therapy. Hum Gene Ther. 2012 [PMC free article] [PubMed]
61. Anderson JS, Javien J, Nolta JA, Bauer G. Preintegration HIV-1 inhibition by a combination lentiviral vector containing a chimeric TRIM5 alpha protein, a CCR5 shRNA, and a TAR decoy. Mol Ther. 2009;17:2103–2114. [PubMed]
62. Walker JE, Chen RX, McGee J, Nacey C, Pollard RB, Abedi M, Bauer G, Nolta JA, Anderson JS. Generation of an HIV-1-Resistant Immune System with CD34+ Hematopoietic Stem Cells Transduced with a Triple-Combination Anti-HIV Lentiviral Vector. J Virol. 2012;86:5719–5729. [PubMed] ** In this study, humanized mice were transplanted with human CD34+ HSCs carrying a triple-agent lentiviral vector. The study confirmed multilineage hematopoiesis of the engineered cells in peripheral blood and various lymphoid organs. A selective advantage of the HIV-resistant CD4+ T cells was observed in mice challenged with either R5-tropic or X4-tropic HIV-1 strains.
63. Kiem HP, Wu RA, Sun G, von Laer D, Rossi JJ, Trobridge GD. Foamy combinatorial anti-HIV vectors with MGMTP140K potently inhibit HIV-1 and SHIV replication and mediate selection in vivo. Gene Ther. 2010;17:37–49. [PubMed] ** A foamy viral vector encodes three anti-HIV genes and the chemotherapeutic-resistant transgene MGMTP140K. Human CD34+ HSCs transduced with the therapeutic vector were engrafted in a humanized mouse and expanded in vivo by drug selection.
64. Burnett JC, Rossi JJ, Tiemann K. Current progress of siRNA/shRNA therapeutics in clinical trials. Biotechnol J. 2011;6:1130–1146. [PMC free article] [PubMed]
65. Abdel-Azim H, Zhu Y, Hollis R, Wang X, Ge S, Hao QL, Smbatyan G, Kohn DB, Rosol M, Crooks GM. Expansion of multipotent and lymphoid-committed human progenitors through intracellular dimerization of Mpl. Blood. 2008;111:4064–4074. [PubMed]
66. Okazuka K, Beard BC, Emery DW, Schwarzwaelder K, Spector MR, Sale GE, von Kalle C, Torok- Storb B, Kiem HP, Blau CA. Long-term regulation of genetically modified primary hematopoietic cells in dogs. Mol Ther. 2011;19:1287–1294. [PubMed]
67. Rossi JJ. Ribozyme therapy for HIV infection. Adv Drug Deliv Rev. 2000;44:71–78. [PubMed]
68. Liu D, Donegan J, Nuovo G, Mitra D, Laurence J. Stable human immunodeficiency virus type 1 (HIV-1) resistance in transformed CD4+ monocytic cells treated with multitargeting HIV-1 antisense sequences incorporated into U1 snRNA. J Virol. 1997;71:4079–4085. [PMC free article] [PubMed]