The Implication of Hinge 1 and Hinge 4 in Micro-Dystrophin Gene Therapy for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by dystrophin deficiency. Dystrophin consists of the amino terminus, central rod domain with 24 spectrin-like repeats and four hinges (H), cysteine-rich domain, and carboxyl terminus. Several highly abbreviated micro-dystrophins (μDys) are currently in clinical trials.

The gRNA Vector Level Determines the Outcome of Systemic AAV CRISPR Therapy for Duchenne Muscular Dystrophy.

Adeno-associated virus (AAV)-mediated clustered regularly interspaced short palindromic repeats (CRISPR) editing holds promise to restore missing dystrophin in Duchenne muscular dystrophy (DMD). Intramuscular coinjection of CRISPR-associated protein 9 (Cas9) and guide RNA (gRNA) vectors resulted in robust dystrophin restoration in short-term studies in the mdx mouse model of DMD. Intriguingly, this strategy failed to yield efficient dystrophin rescue in muscle in a long-term (18-month) systemic injection study.

Rational engineering of a functional CpG-free ITR for AAV gene therapy.

Inverted terminal repeats (ITRs) are the only wild-type components retained in the genome of adeno-associated virus (AAV) vectors. To determine whether ITR modification is a viable approach for AAV vector engineering, we rationally deleted all CpG motifs in the ITR and examined whether CpG elimination compromises AAV-vector production and transduction. Modified ITRs were stable in the plasmid and maintained the CpG-free nature in purified vectors.

Micro-dystrophin AAV Vectors Made by Transient Transfection and Herpesvirus System Are Equally Potent in Treating mdx Mouse Muscle Disease.

Vector production scale-up is a major barrier in systemic adeno-associated virus (AAV) gene therapy. Many scalable manufacturing methods have been developed. However, the potency of the vectors generated by these methods has rarely been compared with vectors made by transient transfection (TT), the most commonly used method in preclinical studies.

Single SERCA2a Therapy Ameliorated Dilated Cardiomyopathy for 18 Months in a Mouse Model of Duchenne Muscular Dystrophy.

Loss of dystrophin leads to Duchenne muscular dystrophy (DMD). A pathogenic feature of DMD is the significant elevation of cytosolic calcium. Supraphysiological calcium triggers protein degradation, membrane damage, and eventually muscle death and dysfunction.

Systemic Delivery of Adeno-Associated Viral Vectors in Mice and Dogs.

Many diseases affect multiple tissues and/or organ systems, or affect tissues that are broadly distributed. For these diseases, an effective gene therapy will require systemic delivery of the therapeutic vector to all affected locations. Adeno-associated virus (AAV) has been used as a gene therapy vector for decades in preclinical studies and human trials.

AAV CRISPR editing rescues cardiac and muscle function for 18 months in dystrophic mice.

Adeno-associated virus-mediated (AAV-mediated) CRISPR editing is a revolutionary approach for treating inherited diseases. Sustained, often life-long mutation correction is required for treating these diseases. Unfortunately, this has never been demonstrated with AAV CRISPR therapy.

The FVB Background Does Not Dramatically Alter the Dystrophic Phenotype of Mdx Mice.

The mdx mouse is the most frequently used animal model for Duchenne muscular dystrophy (DMD), a fatal muscle disease caused by the loss of dystrophin. Mdx mice are naturally occurring dystrophin-null mice on the C57BL/10 (BL10) background. We crossed black mdx to the white FVB background and generated mdx/FVB mice.