Hugh Sweeney

Delivers Micro-dystrophin and Utrophin Chimeric Constructs to the Skeletal and Cardiac Muscles, Optimizing the Gene Therapy to Each Tissue

These micro-dystrophin and utrophin chimeric constructs optimize the treatment of Duchenne Muscular Dystrophy (DMD). DMD is a rare, X-chromosome-linked disorder characterized by progressive muscle degeneration and weakness. Expression of the dystrophin protein is in skeletal, smooth, and cardiac muscle, and mutations in the gene encoding cause DMD. Severe Duchenne cases present total loss of dystrophin from skeletal and cardiac muscles, leading to debilitating muscle degeneration and, ultimately, heart failure. The utrophin protein is highly related to dystrophin and can substitute for dystrophin’s function in mammals. Typically, utrophin is highly expressed in developing muscles and enriches at the neuromuscular junction in mature muscles. As myofibers mature, utrophin levels decrease and express dystrophin.

Gene replacement therapy is the most active and available therapy for DMD. This strategy involves the delivery of a functional dystrophin copy to the patient using viral delivery vectors. However, these vectors have limited carrying capacity, and the large size of dystrophin depicts a compatibility challenge, leading to the need for micro-dystrophin-based gene therapy with a truncated but functional version of dystrophin. While there are ongoing clinical trials implementing these strategies, these therapies are optimized for skeletal muscle without enough evaluation of the clinical impact on cardiac function. This may lead to a form of DMD with severe cardiomyopathy and slowly failing skeletal muscle function as a long-term consequence. Additionally, adverse effects in these clinical trials are present due to neoantigens found in patients with dystrophin isoforms lacking gene regions present in the clinical trial isoforms.

Researchers at the University of Florida have designed micro-dystrophin and utrophin chimeric constructs for treating Duchenne Muscular Dystrophy (DMD) and avoiding micro-dystrophin-associated neoantigen presentation. The utrophin N-terminus replaces the dystrophin N-terminus, reducing the immune response in patients and rescuing the heart and diaphragm muscle without an immune response. The constructs include promoters specific for skeletal or cardiac muscles for enhanced effectivity.

Application

Tissue-specific delivery and expression of micro-dystrophin and utrophin chimeric constructs for the treatment of Duchenne Muscular Dystrophy

Advantages

• Chimeric constructs use tissue-specific promoters, enabling optimized treatment in the skeletal and cardiac muscle tissues
• Chimeric gene constructs replace dystrophin gene regions that can trigger neoantigen presentation with the utrophin equivalent, reducing the potential of undesired immune responses
• Involves catheter-based delivery of gene therapy vectors to the skeletal muscle and cardiac muscle, increasing efficiency and enabling better distribution of the vectors

Technology

These micro-dystrophin and utrophin chimeric nucleic acid constructs provide effective Duchenne Muscular Dystrophy (DMD) treatment. The micro-dystrophin and utrophin sequence combinations enable the replacement of immunogenic micro-dystrophin gene regions with the utrophin equivalent, reducing the risk for neoantigen presentation. Additionally, the constructs have promoters specific to either the skeletal or cardiac muscles and recombinant adeno-associated viral vectors (rAAVs) deliver them to either tissue for targeted or optimized expression of the chimeric proteins in each tissue. More specifically, delivery to a subject consists of a first rAAV comprising a chimeric construct linked to a cardiac muscle-specific promoter to the cardiac muscle and a second rAAV comprising a chimeric construct linked to a skeletal muscle-specific promoter to the skeletal muscle. Delivery of the rAAV particles involves introducing a catheter into the femoral artery and advancing to the heart for delivery of the first rAAV into the coronary arteries (cardiac muscle), and then retracting the catheter to the aortic arch for delivery of the second rAAV to the subclavian and carotid arteries (skeletal muscle). This strategy enables the treatment of each specific tissue independently and efficiently.

Capsid Proteins Optimize Delivery of Therapeutic Genes to Targeted Tissues

These variant recombinant adeno-associated virus (rAAV) particles feature capsid surface proteins that focus delivery of therapeutic genes toward specific tissues, decreasing uptake in non-target tissues. Demand for the AAV gene therapy delivery platform is rapidly increasing as a result of its low pathogenicity and stable transgene expression. Projections value the global gene therapy market at $6.6 billion by 2027 . Gene therapy has many clinical applications for the treatment of rare and terminal human diseases. However, with certain systemic diseases, ensuring the genes get into the right tissues requires a high concentration of AAV vectors, which can cause potentially life-threatening immune responses in patients. Available viral vectors do not adequately target specific tissues.

Researchers at the University of Florida have developed rAAV capsid variants that can increase or decrease uptake of the therapeutic gene particles in a certain tissue of interest. This improved tissue targeting allows for more efficient transgene delivery while reducing the chance of off-target effects.

Application

AAV capsid variants that optimize delivery of gene therapies to target tissues, such as skeletal muscle, central nervous system, or cardiac tissues

Advantages

• Alters the viral protein structure, improving AAV particles’ ability to target specific tissues
• Reduces the amount of virus required, minimizing the chance of undesirable side effects
• Increases efficiency of packed gene therapy particles, decreasing dosages to lower production costs

Technology

These altered rAAV capsids have amino acid substitutions that change their surface protein structure to enhance and/or reduce their tendency toward uptake in certain tissues. This creates a viral vector platform capable of more efficient transduction when delivering therapeutic genes to treat a variety of diseases. Researchers at the University of Florida have tested their variant rAAV particles with altered capsids in skeletal, cardiac, and muscle cells, and have observed improved cell uptake as well as encoded protein production that exceeds that of available synthetic variants.

Combines GDF15 and Myostatin Inhibitor to Treat Obesity While Preserving Muscle Mass

This gene therapy combines the increased expression of GDF15 with a myostatin inhibitor to treat obesity or reduce weight while maintaining muscle mass. Obesity is a major health problem in humans and companion animals. It increases the likelihood of developing diseases such as diabetes, hypertension, coronary heart disease, stroke, and breathing problems, and it correlates directly with mortality risk. Although some available therapies, such as drug therapy and bariatric surgery, do treat obesity, they often cause side effects and other complications, threatening their effectiveness. Previous research has shown the loss of expression of growth differentiation factor 15 (GDF15) correlates with weight gain and worsened metabolic function in mice, making it a potential pathway for treating obesity. However, treatment with GDF15 leads to loss of muscle mass, abating its therapeutic effectiveness.

Researchers at the University of Florida have developed a gene therapy to treat obesity in humans and companion animals with no adverse side effects. By combining the administration of GDF15 and an inhibitor of myostatin, a known negative regulator of muscle growth, this therapy treats obesity while preventing the loss of muscle mass.

Application

Gene therapy combining GDF15 and a myostatin inhibitor to treat obesity in humans and companion animals while preserving muscle mass

Advantages

• Combines administration of GDF15 and a myostatin inhibitor, treating obesity while preventing loss of muscle mass
• When this gene therapy is administered and expressed by obese mice, the mice lose adipose tissue while maintaining lean mass, making it a potential therapy for companion animals and humans

Technology

This gene therapy encodes for the expression of GDF15 combined with a myostatin inhibitor to treat obesity while preserving muscle mass. Loss of GDF15 correlates with weight gain and decreased metabolic function in mice, and previous research has demonstrated that treating mice with GDF15 improves metabolic health in animal models, making it a great potential target for treating obesity. However, although GDF15 decreases adipose tissue volume in mice, it also leads to a dramatic loss in muscle mass through stimulation of myostatin secretion from skeletal muscle, making it a poor therapeutic option on its own. To offset the atrophic effect of GDF15, this therapy combines the administration of GDF15 and an inhibitor of myostatin signaling. To obtain secretion of both GDF15 and the myostatin inhibitor, the inventors have created a bi-cistronic RNA using an internal ribosomal entry site (IRES), with a myostatin propeptide translation following GDF15. Thus, the combination treatment, creating secretion of both GDF15 and a myostatin inhibitor can be used to treat obesity in companion animals and, potentially, in humans. Design alternatives involve administration of GDF15 and the myostatin inhibitor as individual nucleotides, or as polypeptides. Different vectors can serve as delivery vehicles, including adeno-associated viral (AAV) vectors.

Delivers Gene Therapy Specifically to Striated Muscle, Minimizing Systemic Adverse Effects

These recombinant adeno-associated virus (rAAV) capsids enable improved targeting of gene therapy to striated muscle while minimizing delivery to the liver. Duchenne Muscle Disorder (DMD) is a rare genetic disorder involving progressive muscle weakness and degradation due to the defective expression of the dystrophin protein. DMD patients experience initial dystrophy of external muscles, with an eventual impairment of heart and pulmonary function, resulting in a 100 percent fatality rate and a median life expectancy of approximately 28 years. rAAVs have emerged as vectors for the delivery of gene therapy in a number of different diseases, including DMD. However, the majority of delivery strategies involve systemic administration, reducing drug specificity and necessitating high doses, triggering several undesirable immune responses and serious adverse effects.

Researchers at the University of Florida have redesigned rAAV capsid proteins for the targeted delivery of gene therapy to striated muscles while minimizing delivery to the liver. By providing increased tissue-specificity, it enables a reduction in necessary doses, achieving higher efficacy, and reducing costs and safety concerns.

Application

Targeted delivery of gene therapy to striated muscle while minimizing delivery to the liver and adverse immune responses

Advantages

• Provides an increase in striated muscle specificity, minimizing delivery to the liver and undesired side effects
• Reduces the necessary dose required for treatment, decreasing costs, and safety concerns

Technology

The use of recombinant adeno-associated virus (rAAV) has emerged as a therapeutic strategy for the delivery of gene therapy, but most therapies involve systemic administration, leading to adverse immune responses. These modified rAAV present one or more amino acid substitutions in the capsid proteins, leading to surface loops alterations that result in the targeting of receptors important for cardiac and skeletal muscle viral uptake. Consequently, these particles provide improved specificity to striated muscle and higher transduction efficiencies, achieving higher efficacy, minimizing undesired delivery to the liver and reducing costs and safety risks.