Gene Therapy and a Potential Cure for Duchenne Muscular Dystrophy

Introduction to Gene Therapy

Gene therapy is an exciting new form of molecular medicine that introduces a functioning gene into a patient’s cells to compensate for abnormal genes and cure/alleviate the effects of a diseasing-causing mutation. Since its first success in 1990, gene therapy has provided promising new treatments for several genetic diseases such as haemophilia, eye and neurodegenerative disorders and lymphoid cancers in the near future.

Principles of Gene Therapy: Vectors

The objective of gene therapy focuses on transferring the therapeutic gene to its targeted cell, without any form of biodegradation, and ensuring it properly transcribes within the cell.

However, genes that are directly inserted into a cell do not function and therefore must be transferred using a carrier (vector). Bacteria, plasmids and viruses are commonly used due to their small size and efficiency at self-replication. To date, viral vectors (such as adenoviruses, retroviruses, adeno-associated viruses – AAV - and lentiviruses 1) are most often used due to their high transfection efficiency in vivo. In order for viruses to be used as a carrier, scientists must modify the virus using genetic engineering. During this process, the pathogenic parts of the virus are removed and replaced with the therapeutic gene.

Once modified, the vector can either be injected or given intravenously (by IV) directly into a particular tissue and transported to the target cells (in-vivo delivery). Another method includes removing a sample of the patient’s cells and exposing them to the vector before the cells are returned to the patient. Once inside the patient’s cells, the gene is expressed leading to the production of the therapeutic protein and treatment of the disease (ex-vivo delivery).

The Basis of Genetic Diseases

A genetic disease is caused by mutations within the DNA. These abnormalities can range from single-base mutations in one gene to the addition or subtraction of an entire chromosome.

Mutations occur due to a mixture of factors. A likely scenario includes a mutation occurring during DNA replication as the DNA is copied and transferred from one cell to another. This happens approximately every 1 nucleotide out of 104 nucleotides. However, the majority of mutations are corrected through the various DNA repair processes (e.g. proofreading and mismatch repair). This allows new healthy cells containing the correct genetic information to be made during cell division most of the time.

Environmental factors may also play a role in causing mutations. Mutations can be induced from chemicals, such as transposon, “the jumping genes” which bind to the DNA itself and can cause deleterious effects. In addition, abnormalities can be induced by physical mutagens such as radioactive decay, ultraviolet rays and X-rays which alter the DNA bases. Genetic diseases may be inherited if the mutation is present in the germ cells of the parent. Genes in the DNA code for all the proteins (and subsequently essential chemicals such hormones, antibodies, enzymes and haemoglobin). Therefore, a mutation can have disastrous results on the human body and cause genetic diseases to occur.

Genetic diseases are classified into three main types:

Single gene disorders (or Mendelian disorders): caused by mutations in the DNA sequence of a single gene, for instance, cystic fibrosis.

Chromosome disorders: results from changes in the number or the structural arrangement of chromosomes. For example, Down’s syndrome is caused by the addition of an extra chromosome 21.

Multifactorial/Complex: caused by changes in multiple genes, and often due to a complex interaction with environmental and lifestyle factors such as UV rays, cigarette smoke and radiation exposure. Most notable disorders include heart disease, cancer and diabetes.

Duchenne Muscular Dystrophy

All past research and experimentation in developing gene therapies and increased knowledge about genetic epidemiology has provided hope in curing many genetic diseases, including Duchenne Muscular Dystrophy (DMD).

DMD is a lethal neuromuscular disorder, characterized by the progressive deterioration of the muscles. It is caused by mutations that lie within the DMD gene which results to the complete absence of the protein dystrophin in the skeletal muscles. Therefore, the muscle cells become fragile and easily damaged, resulting in an array of other medical complications.

The Duchenne form of muscular dystrophy was named after the French neurologist Guillaume Benjamin Amand Duchenne, who described this condition in his 1861 edition of “Paraplégie hypertrophique de l’enfance de cause cérébrale”. DMD is a Mendelian X-linked recessive disease and therefore occurs predominantly in boys as they do not have another copy of the X-chromosomes to compensate for this genetic defect. However, most girls born with DMD mutations are carriers. Although they are not affected by DMD, carriers can pass DMD genes onto their children. Boys suffering DMD typically present signs as early as 3-5 years of age. Most will depend on a Gower maneuver (push off the floor, lock the knees, then use the hands to “climb up” the legs) to get up from the floor and have difficulty running and climbing the stairs. Children may also experience enlarged calf muscles (pseudohypertrophy) due to abnormal muscle tissue.

Early in the course, muscle mass loss and weakness are observed in the legs, but this eventually spreads to the arms, neck and other areas. Most will require a wheelchair sometime between the ages of 7 to 12. Patients normally suffer too from respiratory failure, pneumonia, or cardiac failure in their twenties and thirties due to weakness of the intercostal and skeletal muscles. Fortunately, ventilation support has been discovered to increase lifespan considerably. Scoliosis and reductions in lung capacity are a serious morbidity in DMD patients, often requiring scoliosis surgery. However, use of glucocorticoids can mitigate scoliosis and the need for spinal surgery. One third of DMD patients may have learning disabilities, attentional disorders, or cognitive delay. Despite the wealth of knowledge, DMD remains an incurable disease which affects as much as 1 in 3500 boys. Much research has been developed within the past century to alleviate the severe symptoms of DMD and increase life expectancy for patients. Unfortunately, many patients only survive to their late teens or early 20s before dying from cardiac or respiratory dysfunction.

The DMD Gene

DMD is caused by a mutation in the 2.4-Mb DMD gene (which encodes dystrophin). Dystrophin is a large intracellular protein that interacts with other proteins at the inner surface of the plasma membrane of muscle cells (myocytes). It is essential for the assembly of these proteins into a transmembrane complex - the dystrophin glycoprotein complex (DGC). This acts as a link between the cytoskeleton and the extracellular matrix in skeletal and cardiac muscles. Because the muscles function by exerting mechanical force on the extracellular matrix, the DGC connection is critical for the transfer of mechanical force which results in movement.

Most mutations which cause DMD are deletions within the coding region of the gene. The majority of these cause frame shifts, leading to premature termination of translation. The absence of the N-terminus of dystrophin prevents the assembly of the DGC, so patients have defective links between their muscle cells and the extracellular matrix. The absence of dystrophin sets in motion hypercontraction of muscle fibers (leading to the enlargement of calf muscles) and eventually the death of myocytes. This results in many of the adverse symptoms present in DMD patients.

History of Gene Therapies for DMD

Despite the early enthusiasm for the development of genetic therapies, several hurdles were presented by the DMD gene. Firstly, gene therapy for DMD requires the delivery of a new dystrophin gene to all of the body’s muscles (which make up greater than 40% of the body mass). Another challenge included the large size of dystrophin gene (2.4 Mb in size), making it incompatible with the current vector models. Immunological responses against viral vectors and newly synthesized dystrophin had been reported as well as difficulty in delivery.

Fortunately, a number of discoveries provided hope for the plausibility of using gene therapy. The isolation of the muscle cDNA which was 14kb but had an 11.2kb open reading frame opened the doors for gene therapy research. This initial observation lead to the discovery that if a synthetic gene derived from this muscle cDNA could be delivered to the myocytes, this would eliminate the need to deliver the entire gene. This enabled the design of a smaller but highly-functional micro-dystrophin as short as 3.6kb.

The development of improved adenoviral vectors also helped overcome many immune-system-related barriers to vector delivery and allowed for delivery of the micro-dystrophin gene. However, these studies were limited to administration via intramuscular injection. Therefore, this approach did not target vital muscles outside of the targeted tissues, such as the heart and respiratory muscles (whose deterioration lead to respiratory and cardiac failure for DMD patients). This discovery paved the way for the synthetization of AAV vectors, which when tested transduced all the striated muscles in adult mice. This led to the demonstration that dystrophy could be almost entirely halted and largely revised in an adult mammal via systemic deliver of AAV vectors containing micro-dystrophin.

Future Prospects

The advancement of gene therapies has provided potential for curing DMD. Gene therapy currently aims at using various vectors to introduce the absent dystrophin gene to a patient’s cells.

Several approaches being contemplated for DMD include the use of AAV vectors to deliver a shorter version of the gene, micro-dystrophin. Various researchers are also testing the possibility of using surrogate gene (for instance, the paralog utrophin, GALGT2, or apha7-integrin) which could partially substitute for dystrophin. Another emerging potential that may be tested in the coming years includes gene editing using the CRISPR/Cas9 system.

The Recent Success of AAVrh74.MHCK7.micro-dystrophin

Sarepta Therapeutics, a medical research company, has recently developed a cure which enhanced dystrophin protein expression in four tested patients. The gene therapy, called AAVrh74.MHCK7.micro-dystrophin uses AAV to deliver micro-dystrophin to the patient’s cells. Although shorter, it contains enough information to produce and restore the function of dystrophin. This gene is designed to target the therapeutic micro-dystrophin gene to the muscles, particularly cardiac muscle, as DMD patients frequently die from heart disease.

The Phase 1/2 clinical trial (NCT03375164) is still ongoing. Each participant involved received an initial single dose of the gene therapy, administered into the blood. Corticosteroids were also taken to prevent immune system responses from targeting and destroying the gene therapy.

Recent data from the four boys showed that the micro-dystrophin gene was successfully delivered to muscle fibres. Muscle biopsies after the delivery showed robust levels of the micro-dystrophin protein, with 81.2% of the fibres being positive for micro-dystrophin. The participants showed improvements in all four parameters established in the North Star Ambulatory Assessment (a scale used to measure functional motor abilities in ambulant children with DMD). If this approach continues to prove successful, this gene therapy may be the cure that scientists and DMD patients have long waited for.

Further Studies Regarding Clinical Trials

For further information about other ongoing clinical trials, please visit the following websites:

Pfizer PF-06939926 Clinical Trial: AAV Micro-dystrophin: https://www.pfizer.com/news/press-release/press-release-detail/pfizer_doses_first_patient_using_investigational_mini_dystrophin_gene_therapy_for_the_treatment_of_duchenne_muscular_dystrophy

Solid Biosciences’ Ignite DMD trial: AAV micro-dystrophin SGT-01: https://www.solidbio.com/about/media/news/letter-to-the-duchenne-community-preliminary-data-and-intention-to-dose-escalate-in-ignite-dmd?fbclid=IwAR3SKt6qPWgjaY1Z_kKMdvIoJW9947mUuGmmLuvI89bZ6BIObvp1MCm4OdY

Conclusions

While DMD was once viewed as a fatal incurable disease that debilitated thousands of individuals and reduced their life expectancy to their late-twenties to early-thirties, gene therapy has provided hope for an effective treatment for DMD. Progression in AAV vector delivery techniques, the synthetization of the micro-dystrophin gene and encouraging early clinical trials suggests that successful gene therapies may soon be available for all DMD patients in the near future.

Acknowledgments

This study was supported and supervised by Laura Finnegan, PhD student at Trinity College Dublin, whom without this would not have been possible.

References

1. Miller AD. Human gene therapy comes of age. Nature. 1992;357(6378):455-460. doi:10.1038/357455a0
2. Nam HY, Park JH, Kim K, Kwon IC, Jeong SY. Lipid-based emulsion system as non-viral gene carriers. Arch Pharm Res. 2009. doi:10.1007/s12272-009-1500-y
3. Munier S, Messai I, Delair T, Verrier B, Ataman-Önal Y. Cationic PLA nanoparticles for DNA delivery: Comparison of three surface polycations for DNA binding, protection and transfection properties. Colloids Surfaces B Biointerfaces. 2005. doi:10.1016/j.colsurfb.2005.05.001
4. U.S. National Library of Medicine. How does gene therapy work? - Genetics Home Reference - NIH. Genetics Home Reference. https://ghr.nlm.nih.gov/primer/therapy/procedures. Published 2018. Accessed March 6, 2019.
5. Baum AS, Garofalo JP. Genetic disorders. In: Encyclopedia of Psychology, Vol. 3. ; 2004:464-466. doi:10.1037/10518-221
6. Pray A, Education PDN. DNA Replication and Causes of Mutation. Nat Educ Knowl. 2008.
7. Pray LA. Transposons: The jumping genes. Nat Educ. 2008.
8. Lee JA. Causes of Mutation. http://www.biochem.uthscsa.edu/med/06-Mechanisms-of-Mutation/PrereqMechanismsofMutation3.html. Published 2015. Accessed March 7, 2019.
9. Kobayashi YM, Campbell KP. Skeletal Muscle Dystrophin-Glycoprotein Complex and Muscular Dystrophy. In: Muscle. ; 2012:935-942. doi:10.1016/b978-0-12-381510-1.00066-1
10. Tyler KL. Origins and early descriptions of “Duchenne muscular dystrophy.” Muscle and Nerve. 2003;28(4):402-422. doi:10.1002/mus.10435
11. Hoffman EP. Dystrophinopathies. In: Rosenberg’s Molecular and Genetic Basis of Neurological and Psychiatric Disease. Academic Press; 2015:1103-1111. doi:10.1016/B978-0-12-410529-4.00091-7
12. Voisin V, De La Porte S. Therapeutic strategies for Duchenne and Becker dystrophies. Int Rev Cytol. 2004;240(SPEC.ISS.):1-30. doi:10.1016/S0074-7696(04)40001-1
13. Hirano K, Sun LT, Tada T. Targeted Chromosome Elimination from ES-Somatic Hybrid Cells. In: Principles of Cloning: Second Edition. Academic Press; 2013:379-389. doi:10.1016/B978-0-12-386541-0.00030-8
14. Sienkiewicz D, Okurowska Zawada B, Paszko Patej G, Kawnik K, Kulak W. Duchenne muscular dystrophy: Current cell therapies. Ther Adv Neurol Disord. 2015;8(4):166-177. doi:10.1177/1756285615586123
15. King TC. Genetic and Perinatal Disease. In: Elsevier’s Integrated Pathology. Mosby; 2016:89-110. doi:10.1016/b978-0-323-04328-1.50010-3
16. Chamberlain JR, Chamberlain JS. Progress toward Gene Therapy for Duchenne Muscular Dystrophy. Mol Ther. 2017;25(5):1125-1131. doi:10.1016/j.ymthe.2017.02.019
17. Braun R, Wang Z, Mack DL, Childers MK. Gene Therapy for Inherited Muscle Diseases. Am J Phys Med Rehabil. 2014;93(11 Suppl 3):S97-S107. doi:10.1097/phm.0000000000000138
18. Inacio P. Sarepta’s DMD Gene Therapy Improves Muscle Function in 4 Boys, Trial Shows. https://musculardystrophynews.com/2018/10/12/sarepta-dmd-gene-therapy-improves-muscle-function-4-boys-trial/. Published 2018. Accessed March 9, 2019.