HIV gets the zinc finger

zinc finger nucleaseBecause all animal viruses initiate infection by binding to a receptor on the cell surface, this step has long been considered a prime target for antiviral therapy. Unfortunately, drugs that block virus attachment to cells have never shown much promise. Another approach, which is to ablate the receptor from the cell surface, is also problematic because these molecules have essential cellular functions. Removing one of the receptors for human immunodeficiency virus type 1 might be an exception.

HIV-1 must interact with two cell surface proteins to initiate infection: a T lymphocyte protein called CD4, and a second receptor, which can be one of two molecules called CCR5 or CXCR4. For many years it has been known that humans can survive without the CCR5 protein: from 4-16% of people of European descent carry the ccr5-delta32 mutation, that prevents the protein from reaching the cell surface. Individuals who are homozygous for ccr5-delta32 (the mutation is present in both copies of the gene) are resistant to HIV infection. Because the vast majority of HIV viruses that are transmitted are those that require CCR5 for cell entry, absence of the protein on the cell surface confers resistance to infection.

The key role of CCR5 in HIV infection in humans was further confirmed when an AIDS patient was given a bone marrow transplant from a donor with the ccr5-delta32 mutation. The patient has been free of HIV for years despite not taking anti-retroviral drugs.

These findings suggest that one possible therapy for AIDS would be to disrupt the ccr5 gene in patient lymphocytes. The development of gene-targeting technologies has brought this approach closer to reality. One approach uses zinc finger nucleases, which are artificial proteins made by joining a protein that can specifically bind DNA with an enzyme that can cleave DNA. A zinc finger nuclease can be designed, for example, to specifically cut within the ccr5 gene. When the cell tries to repair the cut, the gene may be damaged so that the CCR5 protein is no longer made (illustrated).

This approach works: when CD4 T lymphocytes are removed from humans, cultured, and treated with a ccr5 zinc finger nuclease, they become resistant to HIV infection. We discussed this experiment on episode #144 of This Week in Virology.

The next step has now been done: to remove CD4 T lymphocytes from HIV positive donors, treat the cells with the ccr5 zinc finger nuclease (delivered using an adenovirus vector), and infuse the cells back into the patients (each person receives his or her own modified cells). Half of the donors were removed from anti-retroviral therapy, and then the levels of HIV, and CD4 lymphocytes, were measured over the next 250 days.

The result were encouraging: not only were the infusions safe, but the overall levels of CD4 lymphocytes increased, and a good fraction of these had modified ccr5 genes. The initial rise of HIV viremia after interruption of treatment was followed by a decline in virus load. These results show that the CD4 T lymphocytes with modified ccr5 were able to expand in the recipients, and survived better than the unaltered lymphocytes, probably because they were at least partially resistant to HIV infection.

This important clinical trial is only the beginning of a new approach to HIV therapy, and several substantial problems still remain to be solved. Both copies of the ccr5 gene were modified in only 33% of the CD4 T lymphocytes; the remaining cells can still be infected by HIV, albeit less efficiently. New approaches are needed to disrupt both copies of the ccr5 gene in most of the T lymphocytes.

Another issue is that the modified T cells can proliferate for a long time, but not indefinitely. As these cells divide from a limited number of infused cells, they will not have the broad repertoire needed to fight pathogens. T cells are also known to become “exhausted”: they eventually lose their protective functions. Patients given modified lymphocytes still harbor a pool of long-lived T cells which contain HIV DNA. These cells will likely always be present and could give rise to viremia. CD4 T lymphocytes with normal levels of ccr5 protein will always be produced, serving as potential hosts for HIV replication. Modifying stem cells so that they do not produce CCR5 is one long-term solution, but more difficult and dangerous for the patient.

Despite these drawbacks, it is amazing that we can now remove cells from patients, modify their genes, and place them back in patients with little harm and some clear benefit. This is a complicated set of procedures, made even more difficult because humans are involved. It’s truly a landmark clinical trial.

TWiV 204: M m m my corona

On episode #204 of the science show This Week in Virology, Vincent, Alan, Matt and Kathy review isolation of a new coronavirus from two patients in the Middle East, and expansion of the enteric virome during simian AIDS.

You can find TWiV #204 at www.microbe.tv/twiv.

Clinical benefit of lentiviral gene therapy in two patients with a rare neurologic disease

aldX-linked adrenoleukodystrophy (ALD) is a rare neurologic disease caused by a defect in a gene required for normal ABCD1 transporter function. The lack of this function leads to progressive demyelination, severe neurologic disease and death in males, often in childhood. ALD disease progression can be controlled by allogeneic hematopoietic cell transplantation (HCT) in those patients for whom bone marrow donors can be found. This unusual correction occurs because bone marrow-derived monocyte-macrophages are known to migrate into the central nervous system and form functional microglial cells. These corrected microglial cells provide the patients with cells with normal ABCD1 transporter activity and allow normal myelin function.

Two patients with progressive ALD with no available allogeneic HCT donors were recently treated by lentiviral-mediated gene therapy. A lentiviral vector containing the normal ABCD1 transporter gene coding sequence was integrated into the patients’ own marrow derived hematopoietic stem cells after transduction of defective viral particles into the cells ex vivo. The gene-corrected cells were then re-infused into the patients. Myeloablation of the patients’ own marrow was required (as it is in allogeneic HCT treatment) since the gene-corrected cells have no growth advantage over resident patient cells. In addition, the treatment is necessary to provide marrow space for the new gene-corrected cells.

In both of the treated patients, seven and seven and a half years old, there was significant gene transfer and expression of the corrected ABCD1 protein expressed in transplanted myelomonocytes, the population from which the corrected brain glial cells arise. More importantly, brain MRIs from the two patients (left image) showed no progression of demyelinating lesions than in untreated patients (right image). The clinical neurologic status of one patient improved and, in the other, the patient’s disease was stabilized. Untreated patients usually deteriorate in neurologic function when their disease becomes active.

In all HCT gene therapy, stable integration with viruses is required since expansion of specific hematopoietic cell lineages, (here of monocyte-macrophages), are needed to provide adequate numbers of new corrected cells after extensive cell division. Lentiglobin gene transfer (HIV-based) permits integration of transferred genes into quiescent cells (many hematopoietic stem cells are non-dividing) that must be stably transduced for gene therapy with HCT to be successful. By contrast, a property of previously utilized oncoretroviral or gammaretroviral transfer (largely MLV-based) in human gene therapy is that cell division is necessary for viral integration. Consequently gene transfer into HCT is much less efficient.

The clinical benefit provided by this first reported trial of successful lentiviral gene therapy using hematopoietic stem cells in neurologic disease may provide an impetus for similar approaches to treating other neurological diseases.

Cartier N, Hacein-Bey-Abina S, Bartholomae CC, Veres G, Schmidt M, Kutschera I, Vidaud M, Abel U, Dal-Cortivo L, Caccavelli L, Mahlaoui N, Kiermer V, Mittelstaedt D, Bellesme C, Lahlou N, Lefrère F, Blanche S, Audit M, Payen E, Leboulch P, l’Homme B, Bougnères P, Von Kalle C, Fischer A, Cavazzana-Calvo M, & Aubourg P (2009). Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science (New York, N.Y.), 326 (5954), 818-23 PMID: 19892975

TWiV # 12: Prions in milk, lemur lentiviruses, RS virus vaccine, H5N1

twiv_aa_200This Week in Virology #12 has been posted at twiv.tv.

Click the arrow above to play, or right-click to download TWiV #12

In this episode, Vincent, Alan, and Angela discuss Kuru, prions in milk, ancient lentiviruses found in the chromosomes of lemurs, a respiratory syncytial virus vaccine failure in the 1960s, and recent outbreaks of H5N1 influenza in chickens.

If you have subscribed to TWiV via the iTunes store, you have probably found that the podcast is not being updated. Until this issue is sorted out, please re-subscribe to TWiV in iTunes by going to the “Advanced” menu and selecting “Subscribe to Podcast…”. In the window that comes up, paste in the TWiV feed URL:

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