Excision of HIV-1 Proviral DNA from Infected Cells.
An interview of Professor Joachim Hauber, Heinrich Pette Institute - Leibniz Institute for Experimental Virology, Hamburg, Germany.
Antiretroviral therapy (ART) is able to achieve long-term control of HIV-1 replication in a large majority of patients. However, proviral DNA persists for life in some cells, meaning that HIV eradication is not possible with current ART. In order to develop a possible cure, we have to address HIV persistence until the very last infected cells. Dr Hauber and his team are working on a system, named ‘Tre-recombinase’ that could lead to an excision of proviral DNA in these cells. Their recently published paper (1) is for us the occasion to ask further details about this approach
Alain Lafeuillade: Could you explain to us both the principle of this proviral DNA excision molecule and the means used to deliver it into cells and their nucleus?
Joachim Hauber: Tre-recombinase is a Cre-recombinase-derived enzyme that has been engineered in vitro by molecular evolution (2). Tre recognizes and recombines an asymmetric native sequence (of 34 base pairs) in the long terminal repeat (LTR) of a primary HIV-1 isolate, thereby excising the respective HIV-1 proviral DNA from its chromosomal integration site. Thus, Tre-recombinase may be a valuable component of future eradication therapies.
With respect to virus eradication, the delivery of Tre-recombinase most likely requires a gene therapy approach in which either adult CD4+ T cells, or CD34+ hematopoietic stem cells (HSC) are genetically modified using advanced vector systems (e.g. lentiviral self-inactivating vectors) (3). However, particularly the ex vivo treatment of adult CD4+ T cells may also involve the application of cell permeable Tre-recombinase (1). This strategy has the advantage that the genetic modification of HIV target cells by the vector is avoided. The respective recombinant Tre enzyme is characterized by both, a heterologous protein transduction domain (PTD) that ensures transport across the cellular plasma membrane, and a nuclear localization signal (NLS) that mediates nuclear uptake (1).
AL: What are the differences with the approach using Zinc Finger Nucleases (ZFN)? Is there, as for ZFN, a risk of ‘off target’ excision? If yes, is it quantified and what could be the consequences?
JH: By inactivating the cellular HIV coreceptor CCR5, ZFN prevent de novo infection of HIV target cells with CCR5-tropic viruses. In contrast, Tre-recombinase removes HIV-1 from already infected cells, independent of virus tropism (i.e. Tre acts on CCR5-, CXCR4- and dual-tropic viruses). Also in contrast to ZFN, Cre-derived enzymes, such as Tre-recombinase, neither produce free DNA ends (e.g. potentially disastrous double-strand breaks/DBS) nor require additional host factors. Therefore and as we confirmed by state-of-the-art cytogenetic analyses, Tre-mediated recombination is highly accurate and does not produce any genome-wide off-target effects.
AL: In what kind of cells and tissues can you deliver your product? If we move to patients, can such a product be delivered orally?
JH: As indicated above, Tre-expressing vectors will probably be used in the near future in genetic therapies against HIV for transduction of human CD4+ T-cells or, in case of stem cell-based therapies, for genetic modification of CD34+ HSC. Cell permeable Tre-recombinase may become important in the ex vivo treatment of T-cells. However, in the next future oral delivery is not feasible for technical reasons, although we certainly think along this line of development.
AL: Has this kind of approach been already used in humans for other diseases?
JH: Although the direct delivery of cell-penetrating bioactive molecules has been employed in various preclinical models of human diseases, including cancer, psoriasis, and stroke, so far none of these approaches have yet advanced into the clinic for routine application.
AL: If I am right, you think that this approach, in addition to ART could progressively deplete the HIV reservoir. What is the realistic agenda for in vivo development?
JH: We believe that Tre-recombinase will become an important tool in novel HIV eradication strategies. Tre-mediated excision of the proviral DNA in HIV target cells is expected to functionally improve the patient’s immune system, which appears to be critical to the depletion of HIV reservoirs. With respect to virus eradication, it is conceived that Tre-recombinase will be combined with ART and/or purging approaches.
The in vivo development of Tre-recombinase currently concentrates on the analysis of Tre in HIV-infected humanized mice that are transplanted with either Tre-vector transduced CD4+ T-cells or transduced CD34+ HSC. Upon completion of these experiments, clinical studies are envisaged in the near future.
1-Mariyanna L, et al. Excision of HIV-1 Proviral DNA by Recombinant Cell Permeable Tre-Recombinase. PLoS One, 2012: 7, e31576
2-Sarkar I, et al. HIV-1 Proviral DNA Excision Using an Evolved Recombinase. Science, 2007: 316, 1912-15
3-Buchholz & Hauber. In Vitro Evolution and Analysis of HIV-1 LTR-Specific Recombinases. Methods 2011: 53,102-9
Key words: HIV gene therapy, hiv cure, hiv eradication