Administering ZFN and donor gene results in targeted gene replacement.

Scientists claim that genome editing using zinc finger nucleases (ZFNs) and a virally administered donor gene can effectively correct hemostasis in a mouse model of hemophilia. Reporting in Nature, the Childrens’s Hospital of Philadelphia and Howard Hughes Medical Institute team claims ZFNs are able to induce double-stranded DNA breaks efficiently when delivered directly to the mouse liver, and in their studies stimulated gene replacement through both homology-directed and homology-independent targeted gene insertion at the ZFN-specified locus.

The level of gene targeting achieved in the mouse model of hemophilia B was sufficient to correct disease-related clotting times, and persisted after induced liver regeneration. Katherine A. High, M.D., Hojun Li, M.D., and colleagues describe their work in a paper titled “In vivo genome editing restores hemostasis in a mouse model of hemophilia.” The team worked in collaboration with scientists at Sangamo BioSciences.

Gene replacement therapy using viral vector-mediated gene transfer has been successful in animal models and in some human trials, but there are inherent disadvantages with this approach to correcting genetic disorders, the team reports. These drawbacks include potential risks associated with insertional mutagenesis and the loss of endogenous regulatory signals that control gene expression.

An alternative technique for gene therapy involves gene-specific targeting of induced pluripotent stem cells ex vivo and reimplantation of these cells in the patient. However, this method is not applicable to diseases that affect organ systems, such as the liver, for which the ex vivo manipulation of target cells is not feasible.

While ZFNs designed to induce DSBs at specific target loci could provide the answer to gene replacement in vivo, to date it has not been clear whether ZFNs can induce DSBs and stimulate genome editing at a clinically meaningful level in vivo, the researchers continue. To this end, they developed ZFN—and factor IX-carrying hepatotropic viral vectors to test whether ZFN-based gene editing could correct the relevant genetic defect in a mouse model of hemophilia B.

The Children’s Hospital and Howard Hughes team first designed ZFNs targeting the mutated human F9 (hF9) gene, and confirmed their capacity to induce a DSB at the intended target site and stimulate genome editing by homology-directed repair (HDR) in human cells in vitro. They then generated a mouse model (hF9mut/HB) that completely lacks the mouse factor IX gene, but instead expresses a mutated version of the human factor IX protein, resulting in complete absence of circulating factor IX protein.  

Two-day-old hF9mut/HB mice were given intraperitoneal injections of both the AAV-ZFN and AAV-F9 gene constructs, and liver DNA samples were analyzed at 10 weeks to assess gene replacement at the hF9 locus. The results confirmed that HDR-mediated targeting had been effected with about 1–3% targeting efficiency. Circulating human factor IX was also detected in the treated mice, up to about 7% of normal levels. The amount of circulating human factor IX in individual animals correlated directly with detected levels of gene targeting via HDR. Encouragingly, levels of human factor IX in the treated animals persisted after partial hepatectomies.

In a separate series of experiments, the researchers demonstrated that clotting times for animals given the ZFN and donor gene constructs were about the same as for those in wild-type mice, suggesting the treatment had restored hemostasis. They in addition confirmed that most of the expression of human factor IX resulted from specific gene correction, rather than as a result of random integration of the donor gene into the genome. Significantly, treatment appeared to have no growth, or weight-related side effects over eight months, and there were no changes in liver function tests at 4, 29, and 32 weeks after injection, indicating that treatment was well tolerated.

“Together, these data demonstrate a clinically significant correction of the coagulation defect in hemophilia B, via direct in vivo delivery of ZFNs to mediate permanent correction of the genome in mouse hepatocytes,” the authors write.

While translation of the ZFN approach into human gene therapy will require optimization of gene correction efficiency and a thorough analysis of off-target effects in the human genome, the mouse data do provide evidence that AAV-mediated delivery of ZFNs and a donor template give rise to persistent and clinically meaningful levels of genome editing in vivo, “and can thus be an effective strategy for targeted gene disruption or in situ correction of genetic disease in vivo,” they conclude.

“These data represent a significant advance in realizing efficient, systemic, therapeutic gene repair—the holy grail of genetic medicine,” Dr. High adds. “Genome editing also reinstates the wild-type sequence under the control of the endogenous regulatory sequences, assuring restoration of this critical aspect of normal gene expression.”

“This is an important step forward in our goal to broaden the application of ZFN gene-editing via in vivo administration,” states Edward Lanphier, Sangamo’s president and CEO. “These data highlight the therapeutic potential of our ZFN technology and enable us to expand our ZFP Therapeutic pipeline to a growing number of monogenic and rare diseases.”

Sangamo is focused on the development of engineered zinc finger DNA-binding protein transcription factors (ZFP TFs) to up- or down-regulate gene expression, and ZFNs to correct or disrupt genes responsible for monogenic diseases. The firm’s pipeline is headed by the Phase II-stage diabetic nephropathy candidates SB-509, and the HIV/AIDS candidate SB-728.

SB-509 is a ZFP TF designed to up-regulate the gene for vascular endothelial growth factor-A (VEGF-A). In development initially for treating diabetic nephropathy, the candidate is also being evaluated for the potential treatment of amyotrophic lateral sclerosis (ALS), spinal cord and traumatic brain injury and stroke. Phase II trials in ALS have already been conducted.

SB-728 is a ZFN-based approach for modification of the gene encoding CCR5, the major co-receptor used by HIV to infect cells of the immune system, Sangamo notes. The first application of the technology comprises an an autologous ZFN-CCR5-modified T-cell product (SB-728-T), which is being evaluated in Phase I and Phase I/II trials in HIV and AIDS patients. A preclinical-stage program to develop an SB-728 hematopoietic stem-cell (HSC) product and a research-stage program to develop SB-728 as an in vivo product are also in progress.

Sangamo’s earlier-stage pipeline includes zinc finger-based techniques addressing glioblastoma, hemophilia, and rare diseases.

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