More Than One REMEDY for Genetic Disorders

How a new approach to correcting heterozygous mutations and other novel techniques for gene editing are transforming the potential to combat disease

The field of gene therapy has had some astounding success in recent years — much of which emerged from labs at Nationwide Children’s Hospital — but it has also stumbled over some serious logistical roadblocks.

The majority of candidates don’t even make it to clinical trials, often due to practical challenges with development including inaccurate vector delivery, large payload size, inefficient protein production, manufacturing difficulties or inability to administer the therapy to the right cells in the right tissue. Of those that do make it into human trials, off-target effects, inefficacy and unforeseen toxicities have complicated some otherwise promising therapies.

Working With What You’ve Got

Jerry Mendell, MD, advisor to and now the namesake for the Jerry R. Mendell Center for Gene Therapy at Nationwide Children’s, wanted a technology that could distinguish between the good and bad gene in a cell during his time researching therapies for muscular dystrophies. Meisam Naeimi Kararoudi, DVM, PhD, director of the CRISPR/Gene Editing Core at Nationwide Children’s, delivered one better: he created a CRISPR-based technique not just to tell them apart, but to get cells to use the good copy as a template to replace the bad copy.

The technology, dubbed REMEDY, is surpassing even their initial expectations.

“REMEDY became an extraordinarily powerful methodology we could use for a lot of different genetic disorders that already have a good copy of a gene in the cell,” says Dr. Naeimi Kararoudi, who is also a principal investigator in the Center for Childhood Cancer at the Abigail Wexner Research Institute at Nationwide Children’s (AWRI). “It has become the licensing avenue for a lot of our other gene therapy platforms.”

He and his colleagues, including Yasemin Sezgin, MS, research associate and lab manager in the CRISPR/Gene Editing Core, and Allison Bradbury, MS, PhD, principal investigator in the Center for Gene Therapy, have corrected many different mutations, including those causing tubulin folding cofactor D (TBCD)-related developmental and epileptic encephalopathy, cystic fibrosis (CF), Valosin-containing protein (VCP) multisystem proteinopathies (MSP) and numerous other disorders in cell line studies, publishing initial results in Neurology, with more to come.

Afrooz Rashnonejad, MSc, PhD, principal investigator in the Center for Gene Therapy at Nationwide Children’s, has also had success with REMEDY in her laboratory. Her primary research focuses on adeno-associated virus (AAV)-based gene therapies for Charcot-Marie-Tooth (CMT) disease and ACTA1-related congenital myopathy.

“We are using REMEDY in the lab to edit some of the more common mutations that we see in CMT, and it shows great success on induced pluripotent stem cell lines that can later differentiate to Schwann cells, which are the target cells,” Dr. Rashnonejad explains. “We can also package REMEDY components to transfer directly to Schwann cells, and we have successfully edited mutations in the ACTA1 gene using REMEDY.”

Cell line studies have advanced to the point that Dr. Rashnonejad will begin in vivo animal models. The work is funded both by grants and the Nationwide Children’s Technology Development Fund, which helps investigators launch promising studies until they can be supported exclusively by external funding.

More Than One Horse in the Race

Beyond REMEDY, the researchers at Nationwide Children’s are exploring numerous technical approaches to gene therapy.

Another group targeting CMT is the lab of Scott Harper, PhD, principal investigator in the Center for Gene Therapy at Nationwide Children’s and a veteran in the field of gene therapy. Dr. Harper had a prominent role in the design and development of early gene therapies for Duchenne muscular dystrophy, now approved by the Food and Drug Administration (FDA) and marketed as ELEVIDYS. Dr. Harper uses CRISPR to interfere with over-expressed genes that cause CMT and facioscapulohumeral muscular dystrophy (FSHD), namely PMP22 and DUX4, respectively.

“We believe that patients would benefit by reducing these toxic gene levels to a normal range,” says Dr. Harper, now also chief scientific advisor of Armatus Bio, which licensed early techniques from Dr. Harper’s research in the hopes of developing therapies for CMT and FSHD. “We have developed an approach that uses a natural biological process called RNA interference (RNAi) — a cell’s natural way to reduce gene products when they become over-produced. We think it’s superior to CRISPR for this application, and that’s what we’re moving toward in the clinic.”

His lab uses engineered microRNA to prompt cells to degrade the genes’ messenger RNA before they can be used to create toxic levels of proteins.

“Our two lead gene knockdown therapies are in late preclinical stages,” says Dr. Harper. “We’ve demonstrated proof-of-principle and safety in animal models, and are engaging with the FDA with the intention of translating these therapies to clinical trials.”

A Matter of Logistics

Multiple teams in the Center for Gene Therapy, including one led by Zarife Sahenk, MD, PhD, a principal investigator in the center also engaged in CRISPR-based gene therapy for CMT, now share a common goal for advancing these therapies: getting the right vector.

Dr. Zarife’s approach involves a gene therapy paradigm using AAV1.NT-3 to deliver nerve regeneration neurotrophin in CMT animal models.

“The problem is that REMEDY and other CRISPRbased gene therapies are still too big to fit in a single AAV vector,” explains Dr. Naeimi Kararoudi, who together with colleague Nizar Saad, PhD, principal investigator in the Center for Gene Therapy, won the National Institutes of Health Targeted Challenge for the idea of using a novel delivery system for REMEDY to treat progeria. “It is our job to work to make it small enough to fit in one AAV, work with others who can do it, figure out how to use extracellular vesicles for delivery or find a new CRISPR solution so that we can make the therapies as efficient as possible.”

The problem of space is particularly important for researchers working on CF. The CFTR gene at fault for CF is very large and has dozens of mutations that can result in disease. This has led Sriram Vaidyanathan, PhD, principal investigator in the Center for Gene Therapy at Nationwide Children’s, to explore lipid nanoparticles, multi-component delivery and other solutions for packaging a CF gene therapy.

“The transient delivery of lipid nanoparticles and the viral genome-cutting nature of CRISPR/Cas therapies both have potential limitations, which is why we thought single-stranded DNA (ssDNA) may be a good solution,” Dr. Vaidyanathan says. His recently patented technology chemically modifies ssDNA to improve gene insertion and is described in a recent publication in Nucleic Acids Research. “With this method, we can try to edit genes in vivo to optimize both delivery and editing, and improve the potential of gene therapy for mass production.”

After the delivery vehicle is optimized, Dr. Vaidyanathan has yet another challenge to overcome: method of administration. Patients with CF need durable, highly expressed gene correction in multiple organ systems that have high cell turnover.

“When your goal is to make long-lived gene replacement, you are better off targeting stem cell populations,” explains Dr. Vaidyanathan.

He and other researchers in the CF gene therapy community are actively working to explore intravenous and other methods for delivery to the right cell
populations in the lungs to optimize ongoing therapeutic effects of any gene editing.

Left to right (back row): Yasemin Sezgin, Sriram Vaidyanathan, Dean Lee, Meisam Kararoudi, Zarife Sahenk, Afrooz Rashnonejad (front row): Allison Bradbury, Scott Harper.

Down to the Details

Another matter of logistics already partially overcome is how to reduce off-target effects. Many investigators at Nationwide Children’s have so far placed their bets on CRISPR-driven gene editing, and guide techniques that target mutant RNA not found normally in the body.

“It’s challenging to target and drug a transcription factor, which is how the PAX3::FOXO1 fusion oncogene functions in rhabdomyosarcoma, especially when both PAX3 and FOXO1 are also found normally in our body,” says Genevieve Kendall, PhD, principal investigator in the Center for Childhood Cancer at Nationwide Children’s. The PAX3::FOXO1 gene fusion drives these aggressive solid tumors. “If you have significant off-target effects, that’s a problem, because these genes are critical for normal development, and we don’t want treatment to be detrimental.”

When Dr. Kendall began collaborating with Dr. Naeimi Kararoudi to develop therapies for this pediatric sarcoma, they opted to use CRISPR/Cas-13, a newer
version of CRISPR that targets messenger RNA. They have generated and provisionally patented guide sequences unique to the PAX3::FOXO1 gene fusion.

“We have one guide that, when tested in PAX3::FOXO1 rhabdomyosarcoma patient-derived cells, causes the cells to completely stop growing, which was exciting to see,” says Dr. Kendall. “This data made me think this strategy could be impactful. Next, we’ll be testing our CRISPR/Cas13-system in patient-derived xenografts in vivo to optimize delivery and assess efficacy.”

The goal is always to optimize as many parameters as possible before advancing therapies to the next level. That’s why scientists at Nationwide Children’s often test numerous constructs, prioritize use of human transgene models and question the norms about how the science can and should be done.

“In principle, CRISPR approaches could be used to treat both CMT1A and FSHD,” says Dr. Harper. Dr. Rashnonejad, who developed and tested AAV-based Cas13 gene therapy for FSHD, demonstrated its effectiveness in a mouse model. “Although the CRISPR system worked to initially protect mice from DUX4-induced muscle damage, we found that over time, the system stopped working due to an immune response to the bacteria-derived CRISPR proteins,” says Dr. Harper.

The work, currently available as a bioRxiv preprint, will be one of the first to report an immunologic response using CRISPR therapies. To the team, it represents only another technical challenge to overcome — which is standard operating procedure for the folks at Nationwide Children’s.

“Various strategies can be applied to attenuate immune responses to the CRISPR system, including approaches such as engineering Cas proteins to reduce immunogenicity, designing more precise guide RNAs, utilizing tissue-specific promoters and implementing immunomodulation strategies,” explains Dr. Rashnonejad.

Full Steam Ahead

This spirit of innovation, coupled with tremendous intellectual resources and research-centered infrastructure, make Nationwide Children’s the optimal place to advance these gene editing-based therapies, regardless of the selected vector or mechanism of choice of the individual researchers.

“It is really hard to find an institution that has it all — from investigation to manufacturing, clinical teams and a regulatory team, all in one place,” says Dr. Naeimi Kararoudi. “People come to us with an idea and we can do it all for you, with proven successes. There are very few places you can do such a thing.”

In addition to facilitating the work of investigators at Nationwide Children’s and around the world, Dr. Naeimi Kararoudi has had initial triumphs with several of his own projects as well, including gene therapy to combat multiple myeloma and efforts to make therapies more affordable and accessible. His greatest success to date has been with colleague Dean Lee, MD, PhD, director of the Cellular Therapy and Cancer Immunology Program at Nationwide Children’s: an off-the-shelf CRISPR/AAV edited natural killer cell therapy for cancer. The technology received “safe-to-proceed” approval from the FDA to treat cancer patients, a first in many aspects.

And the future looks bright. Gene editing experts at Nationwide Children’s have open minds, robust funding pipelines, savvy technology commercialization allies and a community of veteran gene therapy experts to question and collaborate with on new ideas. Strong relationships with patient advocacy groups and grassroots funding mechanisms stimulate new project ideas and facilitate connections to the patient community.

As company after company finds its roots in Nationwide Children’s intellectual property, the reputation of the Jerry R. Mendell Center for Gene Therapy is only set to grow, providing a rich breeding ground for novel therapies and a brighter future for patients with genetic disorders of all kinds.

References

1. D’Ambrosio E, Ozes-Ak B, Sahenk Z, Mendell J, Kararoudi MN. REMEDY: A Novel CRISPR-based Allele Specific Approach Corrects VCP Mutations (S21. 006). Neurology 2024;102(17_supplement_1: 2426.
2. Kanke KL, Rayner RE, Bozik J, Abel E, Venugopalan A, Suu M, Nouri R, Stack JT, Guo G, Vetter TA, Cormet-Boyaka E, Hester ME, Vaidyanathan S. Single-stranded DNA with internal base modifications mediates highly efficient knock-in in primary cells using CRISPR-Cas9. Nucleic Acids Res. 2024 Dec 11;52(22):13561-13576.
3. Rashnonejad A, Farea M, Amini-Chermahini G, Coulis G, Taylor N, Fowler A, Villalta A, King OD, Harper SQ. Sustained efficacy of CRISPR-Cas13b gene therapy for FSHD is challenged by immune response to Cas13b. bioRxiv preprint, 2 Jan 2025. https://doi.org/10.1101/2024.12.18.629250

Image credits: Nationwide Children’s Hospital