Current HIV/AIDS Reports

, Volume 8, Issue 2, pp 78–84

Gene Therapy Strategies: Can We Eradicate HIV?


    • Infectious Diseases UnitUniversity Medical Center Hamburg-Eppendorf
    • Heinrich-Pette-Institute Leibniz-Institute for Experimental Virology
  • Boris Fehse
    • Research Laboratory for Cell and Gene Therapy, Department for Stem Cell TransplantationUniversity Medical Center Hamburg-Eppendorf
  • Joachim Hauber
    • Heinrich-Pette-Institute Leibniz-Institute for Experimental Virology

DOI: 10.1007/s11904-011-0073-9

Cite this article as:
van Lunzen, J., Fehse, B. & Hauber, J. Curr HIV/AIDS Rep (2011) 8: 78. doi:10.1007/s11904-011-0073-9


Despite the tremendous advances in antiretroviral combination therapy over the last decade, eradication of HIV from the infected organism is still an elusive goal. Lifelong therapy is associated with potential long-term toxicity, adherence problems, and development of drug resistance. Thus, gene therapy approaches targeting viral eradication are still attractive. Here a number of studies have failed to show a clear clinical benefit yet. Current approaches were mainly limited by a low number of transduced cells and genotoxicity. The use of new vector systems and the right choice of target cells and improved transduction protocols may overcome these obstacles. Recent reports on the use of newly developed transgenes either allowing for an enrichment of transduced cells by an in vivo selection advantage or restoration of a functional immune system which is resistant to HIV infection nourished the hope for continuous progress in this field. Indeed the intriguing finding that HIV seems to be eradicated in an individual case study after stem cell transplantation with a mutant coreceptor (CCR5 delta 32 deletion) underlines the proof of the concept.


HIV gene therapyIntracellular immunizationHematopoietic stem cellsGenotoxicityHIV eradication


Since the introduction of highly active antiretroviral therapy (HAART) in the mid-1990s eradication of the virus is still regarded as the Holy Grail by the HIV community. However, complete elimination of HIV has remained an elusive goal. Even patients who are successfully treated with HAART for several years often do not achieve full recovery of immune responses and exhibit increased levels of immune activation along with its detrimental effects. Ongoing low-level viral replication as well as microbial translocation after the disruption of the mucosal barrier and impairment of the mucosa-associated lymphoid tissue (MALT) are thought to be the main drivers of hyper-activation of the immune system in HIV infection now referred to as “immune-aging” or “immunosenescence” [1, 2]. These phenomena are only partially corrected by modern antiretroviral combination therapies. Consequently, viral load rapidly rebounds after HAART cessation since there are inadequate HIV-specific immune responses generated by classical HIV drug therapy regardless of the drug combination used [3]. The preferential infection and subsequent elimination of HIV-specific T-helper cells mainly expressing the CCR5 coreceptor during very early stages of the disease is one major drawback of current antiretroviral therapy approaches. As a consequence, persistence of low-level viral replication as well as the establishment of a pool of latently infected cells might occur, which may severely hamper attempts to eradicate HIV from the infected host by using antiretroviral small-molecule drug approaches alone [4]. Residual viremia may stem from ongoing cycles of viral replication and infection of new cells, in which case further intensification of HAART might be useful. Alternatively, viremia might be the consequence of the release of virions from stable reservoirs of latently infected cells [5]. Since no full cycle of viral replication would be needed for HIV release, intensification would be potentially fruitless in this situation. Almost all of the currently approved antiretroviral drugs target enzymes that interfere with active viral replication and do not affect latent infection. The advent and approval of new drug classes with new modes of action, namely integrase inhibitors and CCR5 antagonists, has revived treatment strategies aimed at eradication of HIV [6, 7]. In the absence of a cure, these strategies may reduce persistent viral load and/or decrease immune activation. However, none of the HAART intensification trials reported so far has shown a significant clinical benefit with regard to viral eradication or downregulation of viral-mediated immune activation [812]. Thus, gene therapy approaches are still an attractive attempt to target residual replication and/or latent infection and to restore immunological properties which are lost early after HIV infection is established.

Clinical Trials Using Genetically Modified Cells

Numerous trials involving the genetic modification of different cell types (T-helper cells, cytotoxic T lymphocytes, peripheral blood stem cells, etc.) by using distinct vector delivery systems have been reported. Early attempts to augment skewed CTL maturation and responses by the adoptive transfer of autologous cytotoxic T lymphocytes have failed to show clinical benefits [13]. Other attempts have been made using peripheral T-helper cells as targets for gene modification [14]. Recently, the first randomized, double-blind, placebo-controlled, phase 2 cell-delivered gene transfer clinical trial was conducted in 74 HIV-1–infected adults who received a tat/vpr-specific anti-HIV ribozyme (OZ1) or placebo delivered in autologous CD34+ hematopoietic progenitor cells. There were no OZ1-related adverse events. There was no statistical difference in viral load between the OZ1 and placebo group at the primary end point (average at weeks 47 and 48), but time-weighted areas under the curve from weeks 40–48 and 40–100 were significantly lower in the OZ1 group. Throughout the 100 weeks, CD4+ lymphocyte counts were higher in the OZ1 group. The results of this study suggest that cell-delivered gene transfer is safe and biologically active in HIV patients [15]. These strategies aimed at an “intracellular immunization” of target cells which are susceptible to HIV infection (eg, T-helper cells, monocytes/macrophages, etc.). However, with regard to eradication of the virus these approaches were only likely to be successful if 1) the transgene confers a selection advantage over non-transduced cells and/or 2) the gene-modified cell is long lived and establishes “immunity to infection” in progeny of their descent. In order to achieve these ambitious goals the choice of the right genetic modification approach will be crucial. There is good evidence to believe that genetic approaches that interfere with viral entry or proviral integration may confer the above-mentioned selection advantage. On the contrary, approaches that interfere at a later step during the viral life cycle are less likely to be successful to this respect [16••].

Particularly the genetic modification of hematopoietic stem and progenitor cells (HSPC), respectively hematopoietic stem cells (HSC), may provide a lasting and sustainable source of HIV-resistant lymphocytes that, over time, may functionally reconstitute the patient’s immune system. The concurrent activation of quiescent viral genomes, for example by transcriptional activation using histone deacetylase (HDAC) inhibitors, may allow purging of latently infected cell pools, which appears to be the most critical step for achieving virus eradication [17].

For the previous two decades various genetic therapies against HIV were clinically evaluated in detail, unfortunately with little success [18]. The failure to achieve detectable antiviral effects in a clinical setting may have at least partly resulted from inadequate gene vector technology and/or inefficient stem cell transduction procedures. It appears that in the near future these technical hurdles can be overcome by new developments such as, for example, self-inactivating (SIN) vector designs, that significantly reduce the risk of insertion mutagenesis (ie, genotoxicity), or increase of HSC transduction rates by inclusion of semen-derived infectivity enhancing fibrils (SEVI) into the transduction protocol [19, 20].

Various inhibitory principles have been analyzed for HIV gene therapy in the past. The respective antiviral genes encoded ribozymes, intracellular single-chain antibodies, transdominant mutants of viral regulatory proteins, cis-active RNA decoy constructs, antisense RNAs, short hairpin RNAs, and membrane-bound peptides that act as fusion inhibitors [18]. Moreover, theoretical modeling suggested that successful antiviral gene therapy depends on a strong antiviral activity (> 30-fold inhibition per replication cycle) that preferably acts prior to the chromosomal integration of the proviral HIV DNA [16••, 21]. However, all of the aforementioned antiviral approaches, irrespective of the fact of whether the antiviral genes acted at the early or late stage of HIV replication, failed to demonstrate antiviral effects in clinical studies, let alone come close to achieving virus eradication.

Proof-of-Concept Studies

The notion that HSC-based gene therapy is a valid concept for the treatment of HIV disease was significantly revived by the case report of the so-called “Berlin patient” [22••]. This HIV-infected patient developed acute myeloid leukemia and therefore underwent repeated allogeneic transplantation with HSC from an HLA-identical donor that was also homozygous with respect to the HIV-1 CCR5 coreceptor inactivating Δ32 allele (CCR5Δ32). Unexpectedly, after transplantation and discontinuation of HAART this patient showed no viral rebound for more than 20 months after cessation of therapy. Very recently the authors published an extended report on the follow-up of this patient 3 years after HSC transplantation. Still they could not detect any relapse of viral replication [23]. Neither virus RNA nor integrated proviral DNA could be detected by the most sensitive methods. The latter is of particular relevance, since prior to allogeneic HSC transplantation also a minor population (2.9%) of CXCR4-tropic and dual-tropic HIV-1 was detected by ultradeep DNA sequencing techniques in this patient. Why these viruses, which should not depend on CCR5 for cell entry, did not rebound after cessation of HAART is currently unknown.

Interestingly, in the present study, a successful reconstitution of CD4+ T cells at the systemic level as well as in the gut mucosal immune system following (CCR5∆32) stem cell transplantation was demonstrated, while the patient remains without any sign of HIV infection. This was observed although recovered CD4+ T cells contain a high proportion of activated memory CD4+ T cells, ie, the preferential targets of HIV, and are susceptible to productive infection with CXCR4-tropic HIV. Furthermore, during the process of immune reconstitution, there was evidence that replacement of long-lived host tissue cells with donor-derived cells took place, indicating that the size of the viral reservoir has been reduced over time. These results strongly suggest that cure of HIV has been achieved in this individual patient.

Clearly, this approach is not applicable to larger patient cohorts for several reasons. For example, prior to HSC transplantation the “Berlin patient” underwent a fully ablative and potentially lethal conditioning regime, which obviously cannot routinely be applied to large numbers of HIV patients. Moreover, chances are extremely rare to identify an HLA-matched and homozygous CCR5-negative HSC donor, which is the very reason why so far no other patient could be treated in the same way. Therefore, a broader application of CCR5-based genetic therapies requires the development of a more direct methodology to generate autologous CCR5-negative cells.

Gene Therapy of HIV: Challenges and Obstacles

As outlined above, HIV infections and AIDS may be viewed as acquired genetic diseases and therefore represent intriguing targets for gene-therapeutic interventions. In fact, a genetic therapy against HIV infection by expressing specific antiviral genes has been suggested as early as 1988 by Friedman and colleagues [24]. Baltimore [25] introduced the term “intracellular immunization” for such strategy. Already in the early 1990s two clinical GT trials were granted by the RAC of the NIH [26]. Also, a gene-marking study in an HIV-discordant identical twin population was initiated at the NIH [13]. Today, approximately 20 clinical trials on gene therapy of HIV infection are listed at the website “” (as of December 03, 2010).

Still, anybody planning a gene therapy study aiming at the eventual eradication of HIV faces a number of questions. Besides selection of the right target gene discussed above, an important question relates to the right target cell. Obviously, this question is directly related to the chosen strategy (above). If the trial aims at eliminating HIV-infected cells by the means of genetically armed cells, lymphocytes or natural killers would have to be gene-modified. On the other hand, the answer is less clear for intracellular immunization approaches. While CD4+ T cells as the main target of HIV seem to be the first choice for intracellular immunization, one might also consider hematopoietic stem cells as the source of all cells infectable by HIV. Both targets have their advantages and drawbacks (Table 1).
Table 1

Potential target cells used for genetic protection from HIV infection

Targets of intracellular immunization

T cells


Easy to obtain



Quantitative reconstitution


In vivo selection required



Ex vivo selection possible



Long-term reconstitution



Loss of present immunity



Risk of insertional mutagenesis

Very limited


Necessity of conditioning



Protection of all potential HIV target cells



While T cells are easy to obtain, their long-term engraftment appears to be limited, at least given the current ex vivo culture conditions. Notably, as taught us the experience with adoptive immunotherapy approaches, conditioning (by suppressing endogenous T cells, ie, lymphodepletion) may strongly improve engraftment of transplanted lymphocytes [27]. However, the actual numbers of T cells which can practically be gene-protected and reinfused are very limited. Consequently, the percentage of “immunized” cells will initially be very low. Any T-cell–based strategy thus will require in vivo selection to become therapeutically relevant. On the other hand, ex vivo protection of high numbers of T cells has the potential to conserve the whole spectrum of T-cell immunity in a given patient. In an early phase of infection this would obviously also include HIV-specific T cells.

On the contrary, hematopoietic stem and progenitor cells have a proven potential of lifelong reconstitution. As shown for inborn severe combined immunodeficiency, a patient has to undergo reduced-intensity conditioning to ensure engraftment of gene-corrected cells [28••]. In principle, it is plausible to transplant selected, “immunized” cells. Thus, all de novo generated T cells (as well as other HIV targets such as monocytes) would be protected against HIV. Alternatively, a stem cell–based approach also had to rely on in vivo selection [29•]. Today’s transplant regimens are much less toxic than those of the 20th century, but still hematopoietic stem cell transplantation is associated with significant risk factors. Moreover, if only “immunized” stem cells are transplanted, the T-helper cell pool present in the patient at the time of therapy will not be protected and therefore be lost if virus replication is ongoing. Consequently, antiviral therapy needs to be maintained until sufficient numbers of gene-protected T cells are generated, which may take years after HSCT, in particular with the heavily aggrieved immune system of HIV patients.

Another relevant question to be addressed relates to the gene transfer method. Since lifelong protection of cells from HIV is aimed for, permanent genetic changes need to be introduced. Based on the current technology, this would in most cases involve integrating gene transfer vectors, preferentially based on γ-retroviral or lentiviral vectors. Both vector types do integrate “semi-randomly,” although with different preferences for different gene regions [30, 31]. It is important to take into account that any method of stable, undirected transgene integration into the genome of the target cell is inevitably associated with insertional mutagenesis (also referred to as “genotoxicity”) [32]. The actual relevance of this risk factor in the context on γ-retroviral gene transfer vectors has been confirmed by a number of severe side effects in clinical gene therapy studies, including myelodysplastic syndromes and leukemia [33•]. At the same time, preclinical data indicate that the risk may be lower for lentiviral as compared to γ-retroviral vectors [34]. Moreover, changes in the vector architecture (eg, SIN-design, use of tissue-specific rather than ubiquitous viral promotors, inclusion of insulator elements) strongly decrease the genotoxic potential of current retroviral vectors, at least in relevant preclinical assays [3538]. However, even with safety-optimized lentiviral vector, insertional mutagenesis cannot be fully excluded [39•]. Importantly, HSC seem to represent a particularly vulnerable cell for insertional oncogenesis [40]. For the selection of the right target cell of anti-HIV gene therapy it is of special relevance that polyclonal mature T cells have been shown to be highly resistant against malignant transformation [41•].

Different strategies are being developed to overcome the limitations of current integrating vectors: If permanent expression of antiviral genes is required, one can either use non-integrating, autonomously replicating vectors to ensure transfer to both daughter cells upon cell division. Alternatively, novel vector systems are being developed which allow direction of the integrating transgene to a “safe harbor,” eg, using the technology of Zink-finger nucleases [42•]. Finally, alternative integrating vectors, for example based on transposons such as “sleeping beauty,” are of great interest [43]. An interesting approach for a permanent genetic modification not requiring long-term transgene expression is the deletion of CCR5. For this modification, short-term expression of specific Zink-finger nucleases in the target cells would be sufficient. Consequently, non-integrating vectors introducing a transient expression of the therapeutic gene would be the tool of choice [44, 45•].

Related to the problem of insertional mutagenesis is the question of how many cells need to be “immunized” to achieve therapeutic efficacy. As has been shown previously, for any integrating vector the number of insertion sites exponentially increases at high gene transfer rates, which would, obviously, lead to a higher risk of genotoxicity [46, 47]. Thus, aiming for very high transduction rates cannot be a reasonable way for currently used retroviral or lentiviral vectors. On the other hand, we know from the so far only effective (natural) gene therapy that infusion of 100% protected cells indeed results in full suppression of HIV replication [22••]. However, genetic modification of all target cells is practically impossible with current technology, even if limited cell numbers are targeted as in a stem cell setting. Therefore, successful outcome relies on selection. In an ablative transplant setting (eg, after conditioning with stem cell toxic drugs), selection may be performed ex vivo before infusion, based on the therapeutic gene approach or a marker gene co-introduced with the therapeutic gene. In that case, almost all infused cells would be “immunized” against HIV. Alternatively, selection may be performed in vivo. To this end, the gene transfer vector might be equipped with a gene encoding a protein that provides a conditional selective advantage, eg, resistance against stem cell toxic drugs [29•]. Alternatively, the strategy may rely on the selective advantage provided by the antiviral gene. This would only be applicable if the antiviral gene protects the cell from cytopathic effects of HIV. As has nicely been shown, for this purpose candidate genes need to tackle early steps in HIV life cycle, namely prevent HIV cell entry or provirus integration [48].

To conclude this part, we are quite close to the introduction of the technical preconditions for performing safe and efficient gene transfer of HIV. To minimize side effects, gene therapy approaches should aim at short-term introduction of genetic material into relevant cells still leading to their permanent genetic modification. This obviously mainly relates to knock-out strategies as eligible for CCR5. Alternative intracellular immunization strategies will soon rely on integration of gene transfer vectors into safe harbors to minimize side effect. Until the underlying technology will be ready for clinical application, γ-retroviral LTR-driven vectors need to be replaced by more advanced, safer vectors utilizing the SIN technology and less trans-activating promotors.

New Concepts

Site-specific nucleases and recombinases provide the technology to manipulate the genome and therefore hold promise for molecular medicine. In particular, zinc finger nucleases (ZFN) are engineered proteins that are composed of a series of linked DNA-binding specificity-providing zinc fingers which are fused to an endonuclease domain [49••]. ZFN cleave as dimers and their DNA-binding domain can be modified to recognize novel target sequences. Therefore, the targeting of two ZFN to selected juxtaposed DNA sites produces a double-stranded break. Its subsequent repair by mutagenic nonhomologous end-joining results in small insertions and deletions that mutate the targeted sequence and thereby frequently inactivate the function of the respective gene product [49••].

Clearly, ZFN would be attractive tools for the directed inactivation of CCR5. In fact, ZFN have been used to disrupt the CCR5 gene in T-cell lines and in primary human CD4+ T cells [50•]. This resulted in impaired surface expression of CCR5 and partial resistance to HIV-1 infection in vitro. Moreover, analysis of HIV-infected mice, which were engrafted with CCR5-negative primary human T cells, demonstrated preferential survival of these engineered cells and substantially lower plasma viremia was observed as in control animals [50•]. These data therefore elegantly demonstrated that CCR5-specific ZFN can be successfully applied for the disruption of the CCR5 gene in human peripheral blood–derived CD4+ T cells.

In a more recent study the ZFN technology was applied for modification of CD34+ HSPC [49••]. Human cord blood–derived HSPC were transiently transfected with plasmids expressing CCR5-specific ZFN, resulting in an estimated 5% to 7% of CCR5−/− cells in the transfected population. ZFN-treated and untreated cells were subsequently transplanted into immunodeficient (NOD/SCID/IL2rγnull) mice, which support human hematopoiesis and HIV-1 infection. Infection of these mice with CCR5-tropic HIV-1 resulted in high levels of viremia and profound CD4+ T-cell loss. In sharp contrast, mice engrafted with ZFN-modified HSPC underwent rapid selection for CCR5−/− cells, had significantly lower viral loads, and CD4+ T cells were not depleted [49••].

This study demonstrated in an impressive manner that only relatively small numbers of HSPC must be genetically modified to achieve significant antiviral effects in vivo. Moreover, well-known potential ZFN-related off-target effects [50•, 51] could be minimized by applying ZFN in a transient manner, thereby apparently avoiding noticeable cytotoxic effects. In general, this therapeutic approach resembles to a large extent the treatment strategy applied in the case of the “Berlin patient.” Thus, directed genetic ex vivo modification of autologous HSPC by ZFN may make therapies based on the inactivation of CCR5 available for the treatment of larger patient cohorts in the future.

It is expected that the ZFN-mediated disruption of the CCR5 gene will neither protect from infection with CXCR4-tropic HIV-1, nor will this strategy be successful in the treatment of patients already harboring significant levels of CXCR4-tropic or dual-tropic viruses. From the point of the clinician, it would be therefore desirable to also have technologies available that are able to literally cure cells from an already established infection. A first approach that succeeded in quantitatively removing integrated HIV-1 proviral DNA in vitro from an infected cell culture is based on the engineering of an HIV-1 long terminal repeat (LTR)–specific recombinase (Tre recombinase) [52••]. Site-specific recombinases, such as the Cre recombinase, recognize and recombine inverted repeat DNA sequences of around 30 to 40 basepairs, which makes these enzymes suitable for engineering of DNA in genomes of heterologous organisms [53, 54]. By applying directed molecular evolution technology, a Tre recombinase had been developed that recognizes a noninverted (ie, asymmetric) native 34 basepair LTR sequence in a primary HIV-1 strain [52••]. Tre-recombinase efficiently recombines this target sequence, which is part of both, the 5′- and 3′-LTR, and thereby excises integrated HIV-1 from human chromosomal DNA. Thus, enzymes such as Tre might be applied in future therapies that aim at virus eradication.

Before Tre recombinase will become a reality in the clinic, various aspects regarding specificity and cytotoxicity have to be carefully evaluated. To name a few, it would be important to identify more conserved LTR sequences, allowing the engineering of a Tre recombinase with extended target range (ie, targeting multiple virus isolates). In addition, such an improved Tre may also prevent the potential problem of outgrowth of a Tre-resistant virus population. Finally, the difficulty of safe delivery of the gene encoding Tre recombinase into human cells has to be resolved.


Taken together, antiviral ZFN and Tre recombinases appear to be important components in the design of future therapies that, for the first time, tackle the problem of HIV eradication with some optimism. It is conceived that both technologies, alone or together, will be combined with intensified HAART regimens or other strategies such as, for example, the activation of latent proviruses. Therefore, new therapeutic approaches that would provide a cure for HIV disease will most likely include HSC-based gene therapies. Technical hurdles with regard to safety (eg, genotoxicity) and transduction efficiency are to be overcome by the development of new vector systems, namely self-inactivating vectors (SIN) which have shown great potential in preclinical studies. Enrichment of protected target cells by a selection advantage in vivo will be crucial to obtain sufficient degrees of gene-marking allowing for a potential cure. In principle, two scenarios are likely to lead to a cure of HIV infection: 1) gene therapy with or without HAART and/or immunomodulation (eg, HDAC inhibitors) will lead to viral eradication; or 2) gene-modified cells will lead to a restoration of immune responses allowing for endogenous long-term control of viral replication which resembles a functional cure.


J. van Lunzen: honoraria from Vision7 GmbH; B. Fehse: stock/stock options in Vision7 GmbH; J. Hauber: none.

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