Publication Spotlight: Long-term multilineage engraftment of autologous genome-edited hematopoietic stem cells in nonhuman primates

Genome editing in hematopoietic stem and progenitor cells (HSPCs) is a promising novel technology for the treatment of many human diseases. Here, we evaluated whether the disruption of the C-C chemokine receptor 5 (CCR5) locus in pigtailed macaque HSPCs by zinc finger nucleases (ZFNs) was feasible. We show that macaque-specific CCR5 ZFNs efficiently induce CCR5 disruption at levels of up to 64% ex vivo, 40% in vivo early posttransplant, and 3% to 5% in long-term repopulating cells over 6 months following HSPC transplant. These genome-edited HSPCs support multilineage engraftment and generate progeny capable of trafficking to secondary tissues including the gut. Using deep sequencing technology, we show that these ZFNs are highly specific for the CCR5 locus in primary cells. Further, we have adapted our clonal tracking methodology to follow individual CCR5 mutant cells over time in vivo, reinforcing that CCR5 gene-edited HSPCs are capable of long-term engraftment. Together, these data demonstrate that genome-edited HSPCs engraft, and contribute to multilineage repopulation after autologous transplantation in a clinically relevant large animal model, an important step toward the development of stem cell-based genome-editing therapies for HIV and potentially other diseases as well.

Publication Spotlight: A curative regimen would decrease HIV prevalence but not HIV incidence unless targeted to an ART-naïve population

HIV curative strategies currently under development aim to eradicate latent provirus, or prevent viral replication, progression to AIDS, and transmission. The impact of implementing curative programs on HIV epidemics has not been considered. We developed a mathematical model of heterosexual HIV transmission to evaluate the independent and synergistic impact of ART, HIV prevention interventions and cure on HIV prevalence and incidence. The basic reproduction number was calculated to study the potential for the epidemic to be eliminated. We explored scenarios with and without the assumption that patients enrolled into HIV cure programs need to be on antiretroviral treatment (ART). In our simulations, curative regimes had limited impact on HIV incidence if only ART patients were eligible for cure. Cure implementation had a significant impact on HIV incidence if ART-untreated patients were enrolled directly into cure programs. Concurrent HIV prevention programs moderately decreased the percent of ART treated or cured patients needed to achieve elimination. We project that widespread implementation of HIV cure would decrease HIV prevalence under all scenarios but would only lower rate of new infections if ART-untreated patients were targeted. Current efforts to identify untreated HIV patients will gain even further relevance upon availability of an HIV cure.

Publication Spotlight: Detection of treatment-resistant infectious HIV after genome-directed antiviral endonuclease therapy

Incurable chronic viral infections are a major cause of morbidity and mortality worldwide. One potential approach to cure persistent viral infections is via the use of targeted endonucleases. Nevertheless, a potential concern for endonuclease-based antiviral therapies is the emergence of treatment resistance. Here we detect for the first time an endonuclease-resistant infectious virus that is found with high frequency after antiviral endonuclease therapy. While testing the activity of HIV pol-specific zinc finger nucleases (ZFNs) alone or in combination with three prime repair exonuclease 2 (Trex2), we identified a treatment-resistant and infectious mutant virus that was derived from a ZFN-mediated disruption of reverse transcriptase (RT). Although gene disruption of HIV protease, RT and integrase could inhibit viral replication, a chance single amino acid insertion within the thumb domain of RT produced a virus that could actively replicate. The endonuclease-resistant virus could replicate in primary CD4(+) T cells, but remained susceptible to treatment with antiretroviral RT inhibitors. When secondary ZFN-derived mutations were introduced into the mutant virus’s RT or integrase domains, replication could be abolished. Our observations suggest that caution should be exercised during endonuclease-based antiviral therapies; however, combination endonuclease therapies may prevent the emergence of resistance.

Publication Spotlight: Safety and Efficacy of Combination Antiretroviral Therapy in Human Immunodeficiency Virus-Infected Adults Undergoing Autologous or Allogeneic Hematopoietic Cell Transplantation for Hematologic Malignancies

The ability to continue combination antiretroviral therapy (cART) in human immunodeficiency virus (HIV)-infected patients undergoing hematopoietic cell transplantation (HCT) for treatment of hematologic malignancies is likely a critical factor in preventing the establishment of an HIV reservoir in transplanted stem cells. Thus, we studied the feasibility of continued antiretroviral therapy in our HIV-infected patients undergoing autologous or allogeneic transplantation. All HIV-infected adults undergoing HCT for hematologic malignancy at Fred Hutchinson Cancer Research Center between 2006 and 2014 were included; most were enrolled in a prospective clinical study to monitor HIV reservoirs after transplantation (NCT00968630 and NCT00112593). Non-nucleotide reverse transcriptase inhibitor or integrase-strand inhibitor-anchored antiretroviral therapy regimens were continued or selected before HCT by infectious disease physicians. Plasma HIV RNA was measured every other day for the first 2 weeks after transplantation and then every 2 weeks. Missed doses of cART and reasons for changing the cART regimen during the post-transplantation hospitalization were documented through review of inpatient pharmacy records. Seven autologous and 8 allogeneic transplantations were performed. In 9 transplantations, the cART regimen was not altered after HCT and no doses were missed. In 2 patients who required alterations in their cART regimen because of development of acute renal failure (n = 1) and small bowel obstruction (n = 1) after HCT, enfuvirtide was used as a bridging component of the regimen. Plasma HIV RNA remained suppressed during the first 28 days in 12 of 15 transplantations, and no patients had a plasma HIV RNA >1000 copies/mL during long-term follow up. Non-nucleotide reverse transcriptase inhibitor- and integrase-strand inhibitor-based cART are safe and effective in HIV-infected persons during the peri-HCT period. Most patients undergoing HCT were able to continue cART without missed doses. Sustained HIV viremia and emergence of resistance were not detected.

Publication Spotlight: Genetically modified hematopoietic stem cell transplantation for HIV-1-infected patients: can we achieve a cure?

The cure of a human immunodeficiency virus (HIV)-1-infected patient following allogeneic transplantation from a CCR5-null donor and potential cure of two patients transplanted with CCR5 wild-type hematopoietic stem cells (HSC) have provided renewed optimism that a potential alternative to conventional antiretroviral therapy (ART) is forthcoming. While allogeneic grafts have thus far suggested complete eradication of viral reservoirs, it has yet to be observed following autologous HSC transplantation. Development of curative autologous transplantation strategies would significantly increase the number of treatable patients, eliminating the need for matched donors and reducing the risks of adverse events. Recent studies suggest gene therapy may provide a mechanism for developing curative therapies. Expression of cellular/artificial restriction factors or disruption of CCR5 has been shown to limit viral replication and provide protection of genetically modified cells. However, significant obstacles remain with regards to the depletion of established viral reservoirs in an autologous transplantation setting devoid of the “allo-effect”. Here, we discuss results from early-stage clinical trials and recent findings in animal models of gene modified HSC transplantation. Finally, we propose innovative combination therapies that may aid in the reduction and/or elimination of viral reservoirs in HIV-1-infected patients and promote the artificial development of a natural controller phenotype.

Publication Spotlight: Combinatorial anti-HIV gene therapy: using a multipronged approach to reach beyond HAART

The ‘Berlin Patient’, who maintains suppressed levels of HIV viremia in the absence of antiretroviral therapy, continues to be a standard bearer in HIV eradication research. However, the unique circumstances surrounding his functional cure are not applicable to most HIV(+) patients. To achieve a functional or sterilizing cure in a greater number of infected individuals worldwide, combinatorial treatments, targeting multiple stages of the viral life cycle, will be essential. Several anti-HIV gene therapy approaches have been explored recently, including disruption of the C-C chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) coreceptor loci in CD4(+) T cells and CD34(+) hematopoietic stem cells. However, less is known about the efficacy of these strategies in patients and more relevant HIV model systems such as non-human primates (NHPs). Combinatorial approaches, including genetic disruption of integrated provirus, functional enhancement of endogenous restriction factors and/or the use of pharmacological adjuvants, could amplify the anti-HIV effects of CCR5/CXCR4 gene disruption. Importantly, delivering gene disruption molecules to genetic sites of interest will likely require optimization on a cell type-by-cell type basis. In this review, we highlight the most promising gene therapy approaches to combat HIV infection, methods to deliver these therapies to hematopoietic cells and emphasize the need to target viral replication pre- and post-entry to mount a suitably robust defense against spreading infection.

Publication Spotlight: Extended Survival of Glioblastoma Patients After Chemoprotective HSC Gene Therapy

Extended Survival of Glioblastoma Patients After Chemoprotective HSC Gene Therapy
Science Translational Medicine
May 2012
Jennifer E. Adair, Brian C. Beard, Grant D. Trobridge, Tobias Neff, Jason K. Rockhill, Daniel L. Silbergeld, Maciej M. Mrugala, and Hans-Peter Kiem

Glioblastoma Patients MRI 1
MRI of the brain (subject one). Axial T1 post gadolinium sequences at the level of midbrain.
A. Area on enhancement seen in the left temporal lobe at the time of initial presentation (arrow). Gross total resection was performed and histology confirmed glioblastoma, WHO grade IV.
B. Area of enhancement and surrounding edema in the left temporal lobe 6 months following diagnosis (radiographically changes were interpreted as possible pseudoprogression). Patient has already received radiotherapy, one dose of conditioning BCNU and stem cell transplantation followed by 2 cycles of temozolomide with O-6-BG when this scan was taken. Patient elected to have surgery and the enhancing abnormality was removed; histology indicated presence of necrosis along with altered, less aggressively appearing tumor. Study drugs were continued.
C. Stable disease with only faint area of linear enhancement seen at the posterior margin of the resection cavity (arrow) 12 month following initial diagnosis (6 months following 2nd craniotomy) after four additional cycles of temozolomide and O-6-BG.

Glioblastoma Patients MRI 2
MRI of the brain (subject two). Axial T1 post gadolinium images.
A. Enhancing lesion in the right parietal lobe (arrow) at the time of initial presentation.
B. First post-radiation scan, enlargement of the enhancing area and associated vasogenic edema (arrow heads) are present (radiographically imaging findings were thought to represent possible pseudoprogression).
C. Six months from the original diagnosis, after 2 cycles of temozolomide and O-6-BG, area of enhancement has enlarged but surrounding edema has diminished and patient was clinically stable.
D. Eleven months from the original diagnosis, after two additional doses of temozolomide with O-6-BG. Area of contrast enhancement is stable.

Chemotherapy is a double-edged sword. Designed to target runaway cells with uncontrolled replication – the hallmark of cancer – healthy cells of the bone marrow, gut, and even hair are also vulnerable because of their intrinsically high replication rates. This collateral damage manifests itself in the form of immunosuppression and intolerable side effects for the patient, often requiring the clinician to reduce the duration and dosage of the chemo regimen as a result. Foreseeably, this alteration of therapy reduces the efficacy of the treatment and further hinders the patient’s chances of recovery.

In this study, defeatHIV co-PI Hans-Peter Kiem of the Fred Hutchinson Cancer Research Center has led a team of researchers in demonstrating the therapeutic potential of gene-modified, chemotherapy-resistant hematopoietic stem cells (HSCs).

Read | Science Translational Medicine commentary: A Shield Against Chemotherapy

What, might you ask, does cancer or chemo-resistant stem cells have to do with our HIV eradication efforts?

Dr. Kiem, along with fellow defeatHIV investigators Drs. Philip Gregory and Michael Holmes, are currently leading a key defeatHIV initiative to develop and test HIV-resistant stem cells in a preclinical model (see descriptions of Projects 2 & 3). Transplant optimization in a preclinical model is essential before clinical testing in people can begin. To this end, generating high levels of the HIV-resistant stem cells following transplant will be an important component of the model optimization. If the HIV-resistant stem cells were also chemo-resistant, this would provide a distinct advantage to their expansion over healthy unmodified cells following chemotherapy.

If we can demonstrate this method of selection for HIV-resistant cells is safe and effective in the preclinical model, we will apply the technique in clinical studies involving HIV-infected individuals requiring bone marrow transplant for hematologic malignancies.

More details on our clinical studies involving HIV-infected subjects are forthcoming. Stay tuned for updates on and in our upcoming Newsletter! Subscribe here.

Read | Extended Survival of Glioblastoma Patients After Chemoprotective HSC Gene Therapy
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Publication Spotlight: Hematopoietic-Stem-Cell-Based Gene Therapy for HIV Disease

Hematopoietic-Stem-Cell-Based Gene Therapy for HIV Disease
Cell Stem Cell
Feb 2012
Hans-Peter Kiem, Keith R. Jerome, Steven G. Deeks, Joseph M. McCune

Investigators from defeatHIV and the Delaney AIDS Research Enterprise (DARE), two of the three NIAID Martin Delaney Collaboratories, partner to review HSC-based gene therapy strategies for HIV disease – demonstrating how scientists in the field are uniting in the fight against HIV.

Read | Hematopoietic-Stem-Cell-Based Gene Therapy for HIV Disease
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