Archive for the ‘Inmunología’ Category

“Berlin Patient” and gene therapy against HIV infection

The case:

Timothy R.B. probably never believed that the same treatment would save him from two different diseases and one of them incurable. He had presented acute myeloid leukemia (FAB M4 subtype) and HIV-1 infection, in the Charité – Universitätsmedizin Berlin (Germany). In the previous 4 years, the patient had been treated with HAART (600 mg of efavirenz, 200 mg of emtricitabine, and 300 mg of tenofovir per day) and no illnesses associated with the acquired immunodeficiency syndrome (AIDS) were observed 1,4,5.

Initial treatment of the acute myeloid leukemia consisted of two courses of induction chemotherapy and one course of consolidation chemotherapy. During the first induction course, severe hepatic toxic effects developed and renal failure occurred. Seven months after presentation, acute myeloid leukemia relapsed, and the patient underwent allogeneic hematopoietic stem-cell transplantation (HSCT) with CD34+ peripheral-blood stem cells from an HLA-identical donor 1,4,5.

Particularly, this donor had been screened for homozygosity for the CCR5 delta32 allele that contains a 32 b.p. deletion, which results in a truncated protein and so generates a non-functional CCR5 receptor phenotype. This chemokine receptor is a cell-surface cofactor for the gp120-CD4 binding, so it is necessary for viral fusion and entry. Some genetic evidence suggests that individuals homozygous for a CCR5 deletion mutation are resistant to HIV infection 1,3,4,5,10,15.

This therapy was described by Eckhard Thiel et al. in “Long-Term Control of HIV by CCR5 Delta32/Delta32 Stem-Cell Transplantation”, on February 12, 2009 in the New England Journal of Medicine 1.

On December 9, 2010 Blood journal pre-published online the article: “Evidence for the cure of HIV infection by CCR5delta32/delta32 stem cell transplantation”, in which the authors showed a reconstitution of CD4+ T cells at the systemic level as well as any sign of HIV infection. The paper quotes: “in conclusion, our results strongly suggest that cure of HIV has been achieved in this patient” 4.

Why gene therapy?

HSCT is used as salvage therapy for patients with malignancies sensitive to chemo-/radiotherapy. These patients receive very dose-intensive cytoreductive regimens designed to eliminate all tumor cells, but the procedure also eliminates the cell substrate necessary to reconstitute the hematopoietic tissue. For this reason it’s require the infusion of HSC (hematopoietic stem cells), that could to reconstitute bone marrow 19.

This therapy presents risks of transplant-related mortality (TRM) due to some complications, as graft-versus-host disease (GvHD), problems derived of inadequate reconstitution of the immune system, the concominant probability of opportunistic infections, and the possibility of malignant relapse. However, the treatment holds great promise as a curative therapy for several tumor types 19, but in the case of HIV infection has insurmountable risks compared with HAART.

Thus, the difficulty in reproducing the therapy is clear, due to the low frequency of the Homozygous Δ32 genotype (1%-3%), the difficulty of finding a matched donor and the problems derived of HSCT. For this reason the application of Gene Therapy seems to be a good option. In this sense I show different approaches that have been made and its possible usefulness in the context of gene therapy against HIV infection.

First of all, I consider myeloablative treatment very important according to the elimination of the reservoirs and infected cells 18, especially having in mind that CCR5 Δ32/Δ32 homozygous does not mean a complete resistance to the HIV infection, due to the presence of CXC4-tropic HIV-1 or dual-tropic. In case of a positive selection of this variant, which normally shows a low frequency (in this patient it was 2.9%) 1,4,5, there are plenty of therapeutic approaches. For instance, the drug AMD3100 8,13 or a siRNA system such the one developed by RJ Pomerantz et al.9, which was quite remarkable. Nevertheless, the importance of this receptor in multiple events (immunomodulation, hematopoiesis, organogenesis and cerebellar neural migration) must be considered 9,10, because inhibition may mean a risk. For this reason, according to its low frequency, the complete reduction of its reservoirs and the stimulation of the Immune System to avoid its reseeding may turn out as a solution 1.

Also, the importance of the anti-HIV myeloablative therapy (although the low success rate raised by I. Huzicka F.18) was shown in two cases in which the DNA/RNA viral detection was negative. It was co-related to an intense pre-transplant cytoablative conditioning (Holland et al. & Contu et al.). The fact of reconstituting the immune system means an opportunity to generate a powerful response to an exhaustive viral burden, similar to the acute infection context 11,12,18. For this reason I think it may be interesting to isolate, stimulate and activate part of the innate immune component (IFN and NK cells) and T cytotoxic of the patient, in order to enhance the innate control events of viremia described in the next image 11,12.


Gene therapy for CCR5 Δ32/Δ32 phenotype generation:

June et al., by using engineered zinc-finger nucleases to disrupt endogenous CCR5 co-receptor in NOG mouse model, generated a robust and stable HIV-resistance genotype, both in vitro and in vivo, which increased the TCD4+ cells recount and reduced viremia (8300 copies/ml) compared with the control (60100 copies/ml). This technology can be designed to make a single break at a specific site in double-stranded genomic DNA, permanently modifying the genome via an imperfect repair by nonhomologous end joining (NHEJ). In this work mice were engrafted with ZFN-modified T CD4+ and showed the previously described results. This technology was also used on human primary T CD4+ cells, obtaining an efficacy and tolerability profile. For this reason an interesting clinic application is proposed 3.

In this context, it may be interesting to consider this technology for autologous CD34+ cells, in order to reconstitute an immune system with phenotype similar to the CCR5 Δ32/Δ32, via autologous stem cells. T cells transfection was accomplished by an adenovirus vector (Ad5/35) that encodes CCR5 ZFNs 3.

Naldini et al. tested the effectivity of the integrase defective lentiviral vectors to allow site specific gen addition through a process which required ZFN break and an homologous DNA template. This approach was carried out, for instance, in CD34+ cells incorporating a GFP cassette at the CCR5 site. Two different experiments showed an addition of the GFP cassete into the CCR5 locus in 80%-90% of the colony-forming cells analyzed 14. Therefore, another possible approach would be the insertion of a transgene in the CCR5 locus, to deplete its expression and thus generate a resistance phenotype to CCR5-tropic HIV-1.

One of the greatest advantages of this system is that it modifies the genotype of the cell without the need of integrating an alien DNA inside the genome since the expression systems used in this work  transiently expressed CCR5 ZFN. According to this, the use of integrative viral vectors which are related to insertional mutagenesis, becomes unnecessary 14,16.

Quoting Naldini et al.’s 14 paper: Gene correction overcomes the major limitations of gene- replacement strategies, as it restores both the function and expression control of the affected gene and avoids the risks associated with semi-random vector integration.

Aubourg et al. implemented a gene therapy in two patients with no matched bone marrow donors. It consisted in isolating CD34+ cells –from peripheral blood mononuclear cells stimulated with granulocyte colony-stimulating factor– by positive selection (with a immunomagnetic technique), and activate them with a mixture of citokines. Then, the cells got infected by replication-defective HIV-1–derived lentiviral vector (CG1711 hALD), which expressed a cDNA of the wild-type ABCD1 gene, which encodes the ALD protein. Transfected cells were then re-infused in both patients after a fully myeloablative conditioning regime with cyclophosphamide and busulfan. The process was clinically uneventful. Also, some months after both patients showed a clinical outcome improvement, similar to the one achieved by allogeneic HCT 2.

I believe that this therapy, through an ex-vivo CCR5 gene alteration, may be very interesting in order to reconstitute an hematopoietic system with a CCR5 Δ32/Δ32 phenotype. Nevertheless, while the aim in the X-linked Adrenoleukodystrophy case was the insertion of a wild-type gene 2, in the VIH resistance case it is necessary just the opposite thing. Hereby the combination of the successful Aubourg et al. approach 2 with the expression of siRNA system against CCR5 expression may turn out useful. Baltimore et al.  constructed a lentivirus-based vector to introduce siRNAs against CCR5 in peripheral blood T lymphocites. This system conferred an important protection against CCR5-tropic HIV-1 infection in these cells 15. However there are problems regarding the integrative viral vectors previously described, as the difficulties with large-scale manufacture in relation to the use of integrative viral vectors in clinical setting 6,17. Before this possible context, nonviral carriers which deliver native siRNA could also be considered, because they generate low immunogenicity and offer high structural and functional tunability 6.

I think all these approximations should be considered, as the continuous progress in correction and gene replacement system, in order to achieve the generation of a HIV resistance phenotype through an autologous HCT. The valuation of the advantages and disadvantages of different systems is necessary though, and particularly its long-term efficacy. For this reason it is also important employing a mouse model with a functional human immune system and an optimal HIV-1 infection profile. Since the major focus of Thiel at al.’ therapy is immune system reconstitution with HIV resistant phenotype, a mouse model which could accept efficient human HSC engraftment should be used. There is a model, the Rag2-/-gammaC-/- double knockout (DKO) mouse, which lacks T, B lymphocytes and NK cells, and in which the injection of CD34+ human HSC directly into the liver of new born DKO mice allows an efficient engraftment, and a reconstitution of the mouse with a functional human immune system in central and peripheral lymphoid organs 7. Using it may be interesting to value the reconstitution with a modified human HSC that expresses CCR5 Δ32/Δ32 phenotype.

Despite advances in gene therapy, this approach is very complicated and therefore in HIV infection would not be a feasible alternative to HAART, actually. However, I consider it an option to develop, especially in cases which HAART is not effective.


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