Enhanced AAV Vectors for Dysferlin Gene Delivery and Evaluation in a Dysferlin Deficient Mouse Model of Muscle Regeneration

Grant Duration
03/10 – 08/16

Objective
Application of Emerging AAV Vector Advancements for the Treatment of Dysferlinopathy.

AAV gene therapy approaches for the treatment of dysferlinopathy have demonstrated efficacy in animal models using dual vectors or fragmented preparations capable of mediating oversized gene transduction. These approaches, while effective and interesting from a basic genetic standpoint, suffer from decreased transduction efficiencies. In particular, our work has demonstrated a concentration dependent effect: there is a larger transduction deficit for a multiple vector approach when the vector genomes are diluted (i.e. in the liver) compared to intact AAV genome transduction (2). Given the likelihood of AAV vector administration via limb perfusion for the treatment of dysferlinopathy, the disseminated target (skeletal muscle), the current AAV production titers, and the host’s immune response to AAV vectors administered at high doses, a complementary strategy using AAV particles containing intact genomes encoding a truncated hybrid dysferlin molecule is under investigation. Current results suggest that this hybrid dysferlin functions in a manner similar to the full length dysferlin and importantly has yet to be associated with toxicity. Our preclinical studies herein seek to offer an AAV viral vector efficient for skeletal muscle transduction with the capacity to restore muscle integrity.

For additional information go to:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3863795/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589059/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5371557/

https://pubmed.ncbi.nlm.nih.gov/26029720/

https://pubmed.ncbi.nlm.nih.gov/28629822/

By Matthew Hirsch, PhD, University of North Carolina (Chapel Hill, NC)|2021-05-07T17:45:21+00:00September 1st, 2016|Past Projects|0 Comments

About the Author: Matthew Hirsch, PhD, University of North Carolina (Chapel Hill, NC)

Dr. Hirsch is an assistant professor in the Department of Ophthamology at the University of North Carolina (Chapel Hill, NC) where he works closely with the laboratory of Jude Samulski at the UNC Gene Therapy Center.

Bla/J mice (B6.A-Dysf^prmd/GeneJ)

Availability:
The Jackson Laboratory
Stock number: 012767
Strain information: http://jaxmice.jax.org/strain/012767.html
Phone: 800-422-MICE (800-422-6423)
610 Main Street, Bar Harbor, Maine 04609 USA

Please note that this strain has been cryopreserved by the Jackson Laboratories, but the Jain Foundation maintains a private colony of these mice for public use. To obtain these mice, please contact the Jain Foundation:

Jain Foundation Inc.
9706 Fourth Ave NE, Suite 101
Seattle, Washington 98115
Phone: 425-882-1492
Fax: 425-882-1050

Email: admin@jain-foundation.org

Development:
In these mice the progressive muscular dystrophy (prmd) allele from the A/J inbred strain is crossed onto the C57BL/6 genetic background. The cross was performed in the laboratory of Dr. Isabelle Richard at Genethon and the backcross generation reached N8. In collaboration with the Jain Foundation, Dr. Richard donated the strain to The Jackson Laboratory in 2010. Upon arrival, mice were bred to C57BL/6J for at least 2 generations to establish the colony.

Mutation:
An ETn retrotransposon (5-6kb) is inserted in intron 4 of the dysferlin gene.

Symptoms:
Disease onset is observed by 2 months and is characterized by the presence of centronucleated fibers and areas of inflammation. As seen with the original background A/J, mice homozygous for the prmd allele on the C57BL/6J background display an increasing number of centronucleated fibers and impairment in the majority of muscles by 4 months of age. In order of severity, the most affected muscles are psoas, quadriceps femoris, tibialis anterior, and gastrocnemius. Mice exhibit a decreased membrane repair capacity following laser wounding experiments. In an open space assay, mice cover less distance and are less active than wild-type. Mice that are homozygous for this allele are viable, fertile and normal in size.

Comparison with other disease strains:
Disease progression is similar to A/J: slightly slower than in SJL/J, Dysf-/- (Campbell), Dysf-/- (Brown), and C57BL/10.SJL mice. As in both SJL/J and Dysf-/- (Brown) mice, proximal muscles are more severely affected than distal muscles. As in Dysf-/- (Brown) mice, abdominal muscles are also affected.

Control strain(s):
C57BL/6 (for homozygotes); littermates (for heterozygotes).

References:
Lostal W; Bartoli M; Bourg N; Roudaut C; Bentaib A; Miyake K; Guerchet N; Fougerousse F; McNeil P; Richard I. 2010. Efficient recovery of dysferlin deficiency by dual adeno-associated vector-mediated gene transfer. Hum Mol Genet 19(10):1897-907.

PMID: 20154340

Below is a quick summary of the inclusion/exclusion criteria. Please review this information carefully prior to making any decision regarding participating.

Inclusion Criteria:

Must be Non-ambulant (cannot walk 10 meters in ≤ 30 sec) and age 18 years or older
Established mutations of the dysferlin gene on both alleles
Impaired muscle function but with sufficient muscle preservation to ensure muscle transfection based on magnetic resonance image of the EDB showing sufficient muscle preservation to permit transfection
Willingness of sexually active subjects with reproductive capacity to practice reliable method of contraception (If appropriate), during the first six months after gene transfer (females) or until two negative sperm samples are obtained post gene transfer (males).

Exclusion Criteria:

Active viral infection based on clinical observations or serological evidence of HIV, or Hepatitis A, B or C infection
The presence of dysferlin mutations without weakness or loss of function
Symptoms or signs of cardiomyopathy, including:
Dyspnea on exertion, pedal edema, shortness of breath upon lying flat, or rales at the base of the lungs
Echocardiogram with ejection fraction below 40%
Diagnosis of (or ongoing treatment for) an autoimmune disease
Persistent leukopenia or leukocytosis (WBC ≤ 3.5 K/µL or ≥ 20.0 K/µL) or an absolute neutrophil count < 1.5K/µL
Concomitant illness or requirement for chronic drug treatment that in the opinion of the PI creates unnecessary risks for gene transfer
Pregnancy
AAVrh74 or AAV8 binding antibody titers > 1:50 as determined by ELISA immunoassay
Abnormal laboratory values in the clinically significant range in the table below, based upon normal values in the Nationwide Children's Hospital Laboratory: GGT, Total Bilirubin, Cystatine, Hemoglobin, White Blood Cells
This first phase of the trial will be small and will only evaluate safety; patients enrolled are not expected to show improvement in their symptoms. If the initial trial is successful, a Phase 2 trial will be scheduled, which will look for efficacy (improvement in symptoms) as well as safety. Each phase would include more patients. So, it is a process and we are pacing ourselves as we support this major milestone in the field of therapeutic intervention for people suffering from dysferlinopathy.