One key to developing a treatment for Huntington’s disease (HD), according to many researchers, involves the challenging task of reducing the expression of the patient’s mutated copy of the huntingtin gene (HTT) while leaving the wild type copy untouched. A collaborative project between Sangamo Therapeutics and the CHDI Foundation, a nonprofit organization exclusively dedicated to developing therapeutics for HD, has developed a pioneering therapeutic strategy that they call “the first direct demonstration of allele-selective transcriptional repression at the native HTT locus.” And, the preclinical data look promising, showing benefits in three different HD mouse models.

“This work is the first demonstration of an engineered, synthetic, modular transcription factor achieving robust allele-selective targeting of endogenous mutant Huntingtin,” notes Adrian Woolfson, PhD, executive vice president of research and development at Sangamo. In addition, he asserts that the novel approach “provides a new method for the field to investigate the role of huntingtin in vivo.”

The work is published today in Nature Medicine in the paper titled “Allele-selective transcriptional repression of mutant HTT for the treatment of Huntington’s disease.” Blair Leavitt, MD, professor in the department of medical genetics at the University of British Columbia who was not involved in the research, tells GEN that it “represents a novel therapeutic approach to HD that selectively targets mutant huntingtin.”

The mutation that causes HD, first described in 1993, consists of repeats of the three base pairs CAG referred to as a “trinucleotide repeat.” These repeats have been a major target for HD drug development over the last 25 years. But, because HTT is involved in other biological functions, and a full knockdown of the gene is lethal in mice, maintaining some expression of the HTT gene is critical.

Now, the researchers at Sangamo have taken a new approach by engineering zinc finger protein transcription factors (ZFP-TFs) to target the CAG repeats in the HTT gene. The team used patient-derived fibroblasts and neurons to demonstrate that the ZFP-TFs selectively repressed over 99% of HD-causing alleles while preserving expression of over 86% of normal alleles.

ZFP-TF technology harnesses the commonly found Zinc-finger class of transcription factors that control gene expression to selectively repress or activate the expression of specific genes. The genes for the ZFP-TF are packaged into AAV vectors which could be administered to a patient to deliver the therapeutic. Once in the cell, the ZFP-TF protein regulates the gene expression of the cell’s genes. Targeting gene regulation differs from other genomic therapeutic approaches, notes Woolfson, because “it is designed to enable precise, robust, and long-term repression of the selected gene following a single administration of AAV and does not cut or modify the target DNA.”

“When we began this project,” notes Bryan Zeitler, PhD, associate director at Sangamo and first author on the paper, “we knew we wanted to screen for allele-specificity at the endogenous HTT locus, but there were no good methods available in the field for monitoring disease versus normal HTT transcript levels independently.” Once the team developed these, and had the right assays in place, they could “rapidly screen for ZFP-TFs with the desired allele-selective property” notes Zeitler.

According to Zeitler, even though they knew that it should be possible to engineer allele-selective ZFP-TFs if they got the designs just right, they were “a bit surprised to find that literally the second ZFP we tested was allele selective.” As they went on to characterize them further, they “were excited to see just how robust the allele-selective repression was across wide dose ranges, multiple patient lines, different cell types, and a variety of disease models.”

How does ZFP-TF compare to other approaches?

This new body of work is particularly exciting for two main reasons according to Sarah Tabrizi, PhD, professor of neurology at the UCL Institute of Neurology and director of the UCL Huntington’s Disease Centre, who was not involved in the work. The first, she notes, is that “it is allele-selective, targeting the mutant HTT gene only, and can potentially treat all patients with HD, unlike allele selective approaches relying on specific SNPs.” Also, Tabrizi asserts that “it targets mutant HTT DNA and prevents transcription, and thereby targets all the potentially toxic downstream forms of the mutant HTT protein.”

Senior author Ignacio Munoz-Sanjuan, PhD, vice president of translational biology at CHDI, agrees, telling GEN that the effects of the ZFPs at the transcriptional level ensure that other mutant HTT products, such as various splice forms of mutant HTT RNA, are also targeted. “In this sense” he adds, “the ZFP repressors are unique in that they eliminate all products of the gene, unlike other therapeutics targeting specific HTT RNA products.”

“Lowering the levels of the toxic mutant HTTby targeting the DNA and RNA and preventing protein expression is the most important area in HD therapies currently,” notes Tabrizi. Another approach to doing this, that has garnered a lot of attention recently, is using antisense oligonucleotides (ASOs.)

Tabrizi was the first author on a recent study published earlier this year in the New England Journal of Medicine that showed promising results using ASO mediated suppression of the HTT protein in the brains of adults with HD. The study, sponsored by Ionis Pharmaceuticals, looked at the effects on their drug IONIS-HTTRx(now RG6042), in 46 patients with early Huntington’s disease.

One reason that the ZFP-TF approach is so promising is that allele-selective ZFP-TFs directly target the cause of the disease—the expanded CAG repeat array in the DNA. “Since all patients have the expanded CAG repeat, the approach is potentially universal,” notes Woolfson.

The delivery method is also a consideration. ASOs are intermittently administered intrathecally, requiring ongoing and life-long visits to a specialized clinical center. “The potential of ZFPs is that they will be administered once directly to the brain via AAV and, provided the drug is sufficiently distributed to the affected brain structures, this one intervention may have profound long-term beneficial effects for patients,” notes Munoz-Sanjuan.

From bench to bedside

“Now the important work needs to happen to see if this promising allele selective gene therapy approach can be translated to patients with Huntington’s disease,” asserts Tabrizi. Indeed, Leavitt notes that “there remain significant unresolved issues related to brain distribution of the agent that will require additional preclinical evaluation before moving to human clinical trials.”

Specifically, Munoz-Sanjuan tells GEN that the major hurdle is “optimizing the ZFP repressor molecules in our study to eliminate or minimize off-target repression of other genes containing CAG repeats, which may pose an unknown risk.” He adds that this has now largely been addressed by Sangamo and the improved ZFP repressors are in late-stage preclinical development.

Sangamo is working with Takeda Pharmaceutical Company to further engineer ZFP-TFs designs to selectively target the mutant HTT gene and repress its transcription. Sangamo also has a collaboration with Pfizer to develop treatments for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), focused on using the ZFP-TF gene regulation approach to repress the expression of the mutated C9ORF72 gene allele linked to these diseases. In addition, they are developing ZFP-TFs to down-regulate the expression of tau, a protein associated with Alzheimer’s disease and other tauopathies.

“The most exciting thing about this work is its unique therapeutic potential,” notes Zeitler. “While we think the science is exciting on its own, our mission is to develop genomic medicines that will make a big difference for patients.” For him, this work is a nice example “of how innovative research can enable novel therapeutic opportunities.”