gene therapy for muscular dystrophy

Gene Therapy for Muscular Dystrophy

By:Mohamed Samir El-Asaly

PT, CKTP

Under Supervision:Porf. Dr.: Mokhtar M. ElZawahri

Introduction The first historical account of

muscular dystrophy appeared in 1830, when Sir Charles Bell wrote an essay about an illness that caused progressive weakness in boys.

Six years later, another scientist reported on two brothers who developed generalized weakness, muscle damage, and replacement of damaged muscle tissue with fat and connective tissue. At that time the symptoms were thought to be signs of tuberculosis.

Introduction In the 1850s, descriptions of boys who

grew progressively weaker, lost the ability to walk, and died at an early age became more prominent in medical journals.

In the following decade, French neurologist Guillaume Duchenne gave a comprehensive account of 13 boys with the most common and severe form of the disease. It soon became evident that the disease had more than one form, and that these diseases affected people of either sex and of all ages.

What is muscular Dystrophy

Muscular dystrophy is a group of diseases that cause progressive weakness and loss of muscle mass.

In muscular dystrophy, abnormal genes (mutations) interfere with the production of proteins needed to form healthy muscle.

There are many different kinds of muscular dystrophy. Symptoms of the most common variety begin in childhood, primarily in boys.

What is muscular Dystrophy

Some people who have muscular dystrophy will eventually lose the ability to walk. Some may have trouble breathing or swallowing.

There is no cure for muscular dystrophy. But medications and therapy can help manage symptoms and slow the course of the disease.

Anatomy of muscle fibers

What is dystrophin? Dystrophin is a protein located

between the sarcolemma and the outermost layer of myofilaments in the muscle fiber (myofiber).

Sacrolemma also called the myolemma, is cell membrane of a striated muscle fibers.

Dystrophin supports muscle fiber strength, and the absence of dystrophin reduces muscle stiffness, increases sarcolemmal deformability and helps to prevent muscle fiber injury.

What is dystrophin??Dystrophin is part of an incredibly complex group of

proteins that allow muscles to work correctly. The protein helps anchor various components within muscle cells together and links them all to the sarcolemma - the outer membrane.

If dystrophin is absent or deformed, this process does not work correctly and disruptions occur in the outer membrane. This weakens the muscles and can also actively damage the muscle cells themselves.

Dystrophin gene The gene for dystrophin

production sits on the X chromosome. If a normal gene for dystrophin is present, then the protein will be made.

If the gene is missing or altered, dystrophin may not be produced at all or only in abnormal forms, resulting in Duchenne muscular dystrophy.

Eukaryote pre-mRNAs often have intervening introns that must be removed during RNA processing (as do some viruses).

intron = non-coding DNA sequences between exons in a gene.

exon = expressed DNA sequences in a gene, code for amino acids.

1993: Richard Roberts (New England Biolabs) & Phillip Sharp (MIT)

Introns and exons

Dystrophin gene biology The X-linked dystrophin gene (DMD) is by far the largest of

the 19,000 genes that encode proteins in the human genome. Its 79 exons cover 2.6 million base pairs (bp). This large size makes the gene prone to rearrangement and

recombination events that cause mutations. In most cases, the mutations are deletions of one or more

exons. In general mutations that disrupt the reading frame of the

dystrophin transcript and lead to prematurely aborted dystrophin synthesis cause Duchenne muscular dystrophy (DMD).

Dystrophin gene

Types of Muscular Dystrophy

Duchenne

Becker MD

Congenital MD

Emery-Dreifuss MD

Distal MDFacioscapulohumera

l MD (FSHD)

Myotonic dystrophy

(DM1)

Limb-girdle MD (LGMD)

Oculopharyngeal MD (OPMD)

Duchenne MDIs the most common childhood form of

MD, accounting for approximately 50 percent of all cases. Because inheritance is X-linked recessive (caused by a mutation on the X, or sex chromosome)

Duchenne MD primarily affects boys, although girls and women who carry the defective gene may show some symptoms.

Duchenne Muscular Dystrophy Inheritance DMD is inherited in an recessive

pattern (defect at Xp21 locus) Females will typically be carriers

for the disease while males will be affected

The son of a carrier mother has a 50% chance of inheriting the defective gene from his mother.

The daughter of a carrier mother has a 50% chance of being a carrier or having two normal copies of the gene

Prevalence of DMD Affects one in 3500 to 5000 newborn males 1/3 of these with previous family history 2/3 sporadic There are two groups that make up the

sporadic cases. ½ of the sporadic cases, there was a genetic mutation in the mother’s egg or a genetic mutation early in embryo development that led to the condition.

In the other ½ of sporadic cases, the mother is a carrier, but she is carrying a new mutation that occurred in either her mother’s egg, her father’s sperm, or early in her development.

This explains the cases where a boy is born with Duchenne muscular dystrophy into a family with absolutely no history of the condition

Initial Symptoms of DMD Delayed developmental milestones A waddling gait Pain and stiffness in the muscles Difficulty with running and jumping Walking on toes Particularly large calf muscles Difficulty sitting up or standing Learning disabilities, such as

developing speech later than usual Frequent falls.

Later symptoms of DMD Inability to walk

A shortening of muscles and tendons, further limiting movement.

Breathing problems can become so severe that assisted breathing is necessary.

Curvature of the spine can be caused if muscles are not strong enough to support its structure.

The muscles of the heart can be weakened, leading to cardiac problems.

Difficulty swallowing; this can cause aspiration pneumonia and a feeding tube is sometimes necessary.

Signs of DMD Has a hard time lifting his head or has a

weak neck Has a hard time walking, running, or climbing

stairs Is not speaking as well as other kids his age Needs help getting up from the floor or walks

his hands up his legs in order to stand (see Gower Maneuver, right)

Has calves that look bigger than normal (pseudohypertophy)

Walks on his toes and waddles Walks with his chest pointed out.

Becker MD Is less severe than but closely related to

Duchenne MD.People with Becker MD have partial but

insufficient function of the protein dystrophin.There is greater variability in the clinical course

of Becker MD compared to Duchenne MD.The disorder usually appears around age 11 but

may occur as late as age 25, and affected individuals generally live into middle age or later.

Becker MD The rate of progressive, symmetric (on both sides

of the body) muscle atrophy and weakness varies greatly among affected individuals.

Many individuals are able to walk until they are in their mid-thirties or later, while others are unable to walk past their teens.

Some affected individuals never need to use a wheelchair. As in Duchenne MD, muscle weakness in Becker MD is typically noticed first in the upper arms and shoulders, upper legs, and pelvis.

Symptoms Becker MDEarly symptoms of Becker MD include: walking on one's toes. Frequent falls, and difficulty rising from the floor. Calf muscles may appear large and healthy as

deteriorating muscle fibers are replaced by fat, and muscle activity may cause cramps in some people.

Cardiac complications are not as consistently present in Becker MD compared to Duchenne MD, but may be as severe in some cases.

Cognitive and behavioral impairments are not as common or severe as in Duchenne MD, but they do occur.

Becker MDMutations in the dystrophin gene that do not

disrupt the translational reading frame result in the milder Becker muscular dystrophy (BMD) phenotype, which is found in 1 in every 20,000 newborn boys.

BMD patients therefore show intermediate to mild phenotypes and have much longer life expectancies.

How are the muscular dystrophies diagnosed? Individual's medical history. Complete family history. It is also important to rule out any

muscle weakness. Thorough clinical and neurological

exams can rule out disorders of the central and/or peripheral nervous systems, identify any patterns of muscle weakness and atrophy, test reflex responses and coordination, and look for contractions.

Muscular Dystrophy Diagnosis

Exercise testsNerve conduction velocity studiesElectromyography (EMG)Electron microscopyMuscle biopsies

Blood and urine testsCreatine kinase is an enzyme that leaks out of

damaged muscle. Elevated creatine kinase levels may indicate

muscle damage, including some forms of MD, before physical symptoms become apparent.

Levels are significantly increased in the early stages of Duchenne and Becker MD.

Testing can also determine if a young woman is a carrier of the disorder.

Blood and urine testsMyoglobin is measured when injury or

disease in skeletal muscle is suspected. Myoglobin is an oxygen-binding protein

found in cardiac and skeletal muscle cells.

High blood levels of myoglobin are found in people with MD.

Blood and urine testsThe level of serum aldolase, an enzyme

involved in the breakdown of glucose, is measured to confirm a diagnosis of skeletal muscle disease.

High levels of the enzyme, which is present in most body tissues, are noted in people with MD and some forms of myopathy.

Diagnostic imaging Magnetic Resonance

Imaging (MRI), is used to examine muscle quality, any atrophy or abnormalities in size, and fatty replacement of muscle tissue, as well as to monitor disease progression.

Ultrasound imaging (also known as sonography), Ultrasound may be used to measure muscle bulk.

Immunofluorescence Testing can detect specific

proteins such as dystrophin within muscle fibers. Following biopsy, fluorescent markers are used to stain the sample that has the protein of interest.

In this example: Dystrophin IMF1. Normal: Localized to myocyte

membrane.2. BMD: Present but reduced.3. DMD: Completely absent

Genetic counseling Two tests can be used to help expectant parents find out if their

child is affected.1. Amniocentesis, done usually at 14-16 weeks of pregnancy, tests a

sample of the amniotic fluid in the womb for genetic defects (the fluid and the fetus have the same DNA). Under local anesthesia, a thin needle is inserted through the woman's abdomen and into the womb. About 20 milliliters of fluid (roughly 4 teaspoons) is withdrawn and sent to a lab for evaluation. Test results often take 1-2 weeks.

Genetic counseling2. Chorionic villus sampling, or CVS, involves the removal and

testing of a very small sample of the placenta during early pregnancy. The sample, which contains the same DNA as the fetus, is removed by catheter or a fine needle inserted through the cervix or by a fine needle inserted through the abdomen. The tissue is tested for genetic changes identified in an affected family member. Results are usually available within 2 weeks.

Multiplex PCRThe most basic method

still in regular use involves multiplex PCR of the exons known to be most commonly deleted.

This method was first published by Chamberlain et al. in 1988.

Analysis of dystrophin gene by multiplex PCR. I: patient with deletion of exon 45, II: patient with deletion of exon 48, and III: normal control.

Multiplex PCRThe advantage of this method is its

relative simplicity.However it does not detect

duplications, does not characterize all deletion breakpoints, and cannot be used for carrier testing of females.

Quantitative analysisQuantitative analysis of all exons of the gene

have brought about an improvement in mutation detection rate, as they will detect all exon scale deletions as well as duplications.

Able to detect mutations in carrier females.Of the quantitative methods available, multiplex

ligation-dependent probe amplification (MLPA – a commercial kit developed by MRC-Holland) is now the most widely used.

Quantitative analysisUse of oligonucleotide-based array comparative

genomic hybridisation (array-CGH).This method analyses copy number variation

across the entire gene. Has the added advantages of detecting complex

rearrangements and large scale intronic alterations and delineating mutation break-points much more closely.

Full Sequence AnalysisIf no deletion or duplication is detected, then, in

the case of DMD patients, full sequence analysis should be undertaken.

Sequencing can be carried out on either genomic DNA or muscle-derived cDNA.

Analysis of genomic DNA has the advantage that it does not require the patient to undergo a muscle biopsy.

Full Sequence AnalysisAnalysis of genomic DNA will not detect

mutations in the 2% of cases with complex rearrangements or deep intronic changes.

Analysis of muscle RNA therefore has a slightly higher sensitivity, and is more amenable to laboratories with less automation, however the requirement for a muscle biopsy is a drawback.

Treatments for DMDTo improve

breathing:O2 therapy

Ventilator

Scoliosis surgery

Tracheotomy

Treatments for DMDTo improve mobility:

Physical therapy

Surgery on tight joints

Prednisone

Non-steroidal medications

Wheelchair

Dystrophin gene Dystrophin consists of 4 main

domain:1. The N-terminal domain (red)

binds to F-ACTIN.2. The cysteine-rich domain (green)

binds to β-DYSTROGLYCAN (β-DG).3. The C-terminal domain (yellow)

binds to DYSTROBREVINS and SYNTROPHINS.

4. The central coiled-coil rod domain (blue) contains 24 SPECTRIN-like repeats (R1–R24) and 4 ‘hinge’ regions

Mini- and Micro-dystrophins

Reduction of the dystrophin transgene size.A large range of deletions in dystrophin cause

only mild phenotypes in BMD patients.So, large parts of the gene seem not to be vital

for function. To map the regions that are crucial for

dystrophin function, several transgenic mice were engineered to carry different deletions throughout the four dystrophin domains.

Mini- and Micro-dystrophins

Deletions in the N-terminal domain were associated with relatively mild phenotypes, which indicates that this region might be important but not essential for attachment to actin and the cytoskeleton.

By contrast, deletions in the cysteine-rich domain cause severe dystrophy, owing to disruption of the entire dystrophin–glycoprotein complex.

The C-terminal domain, with its various alternative splicing patterns, seems not to be required for the assembly of this complex.

A series of large deletions that were evaluated in the central rod domain indicated that although the rod structure is indispensable, the number of repeats can be markedly reduced.

Mini-dystrophins A 6.2 kb mini-construct that contained 8 repeats and

hinge regions 1, 3 and 4, was engineered to mimic the exon 17–48 deletion in a BMD patient described by England and colleagues.

This construct was found to be completely functional: transgenic mice that carried this construct showed non-dystrophic muscle morphology and normal force generation.

Micro-dystrophins Further reduction is feasible, as shown by several micro-

constructs (3.6–4.2 kb) that were highly effective in supporting almost normal muscle structure and function, at least in mice.

The smallest effective micro-construct was only 3.6 kb in size and carried 4 repeats and hinge regions 1, 2 and 4.

Gene Therapy StrategiesGene augmentation approaches deliver

functional cDNA, such as micro- dystrophin or micro-utrophin cDNA, to compensate the function of dystrophin protein.

Retroviral vectors or DNA transposonvectors stably integrate the therapeutic cDNA into chromosomes randomly.

Due to the large size of the dystrophin or utrophin cDNA, transduction efficiency is one of the biggest obstacles.

Gene Therapy Strategies

Genome editing approaches modify the mutated gene specifically, but off-target mutagenesis is a concern. The delivery of programmable nucleases is unexplored in the context of muscle tissue.

Exon skipping uses antisense oligonucleotides to modulate the splicing patterns of a particular exon. Systematic delivery is feasible for antisense oligonucleotides, but the effect is transient.

Risk of off-target effects or posttranslational suppression of the target gene should also be considered.

Gene Replacement Therapy

To replace a defective dystrophin gene, an artificial dystrophin cDNA construct must be transferred into the nuclei of muscle cells, where it must be expressed and regulated appropriately.

So, to deliver the 14 kb dystrophin cDNA vectors with a large capacity were needed.

The capacity of first generation adenoviral vectors (up to 8 kb) was too small.

Later, high capacity (28 kb) ‘gutless’ vectors, from which all adenoviral genes had been removed, bypassed this restriction and delivered extra benefits in the form of reduced host immune response to the viral vector and improved persistence of transgene expression in muscle.


However, two crucial problems need to be overcome before adenoviral vectors can be used therapeutically:

1. They are too large to easily cross the EXTRACELLULAR MATRIX that surrounds mature myofibres

2. There are not many adenoviral attachment receptors on the surface of myofibres


In contrast to adenoviral vectors, herpes simplex virus type-1 (HSV-1) vectors can naturally carry large inserts.

HSV-1 vectors have shown relatively high TRANSDUCTION levels in vivo, but, similar to gutless adenoviral vectors, this is only seen in newborn and regenerating muscle.

The IMMUNOGENICITY and CYTOTOXICITY of HSV-1 hampers the long-term expression of transgenes.


The size of the full-length dystrophin cDNA is not a problem for non-viral DNA plasmid vectors that can be engineered to contain large inserts.

These vectors are synthetic and non-infectious, so they are highly suitable for use.

This delivery strategy is inefficient in muscle tissue, so other strategies are needed to enhance TRANSFECTION efficiencies. Less invasive, and preferably systemic, methods are needed before plasmid vectors can be used.

Transgene delivery with rAAV vectors

Transgenes (for example, the mini- or micro-dystrophins) are cloned in between the AAV inverted terminal repeat (ITR) sequences, under the control of a promoter of choice (for example: CMV, MCK or CK6).


Different mini- and micro-dystrophin gene constructs were cloned into rAAV type-2 vectors and tested in md mice.

These studies showed that the rAAV delivery of constructs carrying four, five or eight repeats, in combination with either two or three hinge regions, was an effective means of treating DMD symptoms in this model.

Reversal of the md-associated morphological abnormalities was observed up to at least six months post-injection, independent of the choice of promoter (CMV, MCK or CK6), the injected muscle (gastrocnemius or tibialis anterior) and the age of the mice at the time of injection.

Overall, there was widespread high expression of the mini- and micro-dystrophins, with correct localization at the fibre membranes and a restored dystrophin– glycoprotein complex.


The therapeutic effect was characterized by the correction of several pathophysiological parameters that are associated with the md mice phenotype, such as variable fibre diameters, myofibres with central nuclei, reduced membrane integrity and necrosis.

These data indicate that the rAAV-mediated delivery of effective mini- or micro-dystrophin gene constructs can slow or even halt the progression of the dystrophy on a long-term basis.


However, a greater immune response against rAAV delivered (foreign) transgene products was observed in dystrophic muscle compared with normal muscle. This was attributed to the inflammatory md-muscle environment and the effect of co-infecting antigen presenting cells (APCs) that activate cytotoxic T lymphocytes against the transgenic products, which causes the destruction of transduced myofibres.

To minimize the immune responses in rAAV-based DMD gene-therapy studies, it will be necessary to use immunosuppressing drugs, muscle-specific promoters to avoid the activation of APCs and fully functional micro and mini-dystrophin constructs to protect fibres from degeneration and the release of neo-antigens.

Antisense-Induced Exon Skipping

Antisense oligodeoxyribonucleotide is a short single stranded nucleic acid, typically 15-25 nucleotides in length, that has the ability to mediate theraputic effects by directly interacting with pre-mRNA or mRNA in a sequence specific manner.

Theraputic antisense are normally designed to bind to relevant exon-intron junction in thr pre-mRNA; blocking of splicing at the junction may induce skipping of an adjucent exon containing the harmful mutation.


The relatively mild BMD phenotypes that are caused by some large deletions or nonsense mutations have also pointed to another possible gene-therapy strategy:

Skipping an exon during PRE-mRNA SPLICING to enlarge a DMD deletion so that it becomes its nearest in-frame BMD counterpart.

So, it was clear that skipping exons could have some therapeutic value, but the question remained: how could this be artificially induced?


The answer came from a study on a Japanese DMD patient, in which a 52-bp frame-disrupting deletion in exon 19 was found to cause exon 19 skipping from the dystrophin transcript.

It was proposed that this region might contain an exon recognition site (ERS) — also known as an exon-splicing enhancer (ESE) — which is a purine-rich sequence that is required for the correct splicing of exons with weak splice-site consensus sequences.

A small antisense oligodeoxyribonucleotide (ODN) was tested and found to block this ERS sequence, as judged from the precise skipping of exon 19 following transfection of this ODN into human lymphoblastoid cells.


The skipping of only 12 different exons could theoretically correct almost 75% of all deletions.

CRISPR-Cas9CRISPR-Cas9 is a unique technology that

enables geneticists and medical researchers to edit parts of the genome by removing, adding or altering sections of the DNA Sequence.

It is currently the simplest, most versatile and precise method of genetic manipulation and is therefore causing a buzz in the science world.

It can be used in a point mutation in the dystrophin gene.

CRISPR-Cas9 The CRISPR-Cas9 system consists of two key molecules that

introduce a change mutation into the DNA. These are: An enzyme called Cas9. This acts as a pair of ‘molecular

scissors’ that can cut the two strands of DNA at a specific location in the genome so that bits of DNA can then be added or removed.

A piece of RNA called guide RNA (gRNA). This consists of a small piece of pre-designed RNA sequence (about 20 bases long) located within a longer RNA scaffold. The scaffold part binds to DNA and the pre-designed sequence ‘guides’ Cas9 to the right part of the genome. This makes sure that the Cas9 enzyme cuts at the right point in the genome.

Utrophin Upregulation Dystrophin has a homologue called utrophin. The

utrophin gene (UTRN) maps to chromosome 6q24 and contains.

Although the total genomic length of the utrophin gene is only approximately one-third of that of the dystrophin gene, its transcript is (at 13 kb) almost as large. Dystrophin and utrophin are highly similar and one probably originated from a duplication of the other.

The most prominent difference is that it lacks the spectrin-like repeats 15 and 19, and 2 hinge regions of dystrophin.

Utrophin Upregulation Utrophin is ubiquitously expressed in most

tissues— most prominently in lungs, blood vessels and the nervous system.

In muscle, its local expression is maturation dependent: in fetal muscle it is initially dispersed over the SARCOLEMMA, during development it is gradually replaced by dystrophin and in mature muscle it is located only at the neuromuscular and myotendinous junctions.

At the post-synaptic membrane of the neuromuscular junctions, utrophin co-localizes with the ACETYLCHOLINE receptors and is thought to have a structural and functional role in the differentiation and maintenance of postsynaptic membrane domains.

Utrophin UpregulationBy contrast, in the regenerating muscle of DMD patients,

mdx mice and dystrophin-deficient cats, utrophin was found to be both upregulated and redistributed to the sarcolemma.

This latter observation led to the hypothesis that utrophin might have a complementary, as well as a protective, role in dystrophic muscle.

Further support for this hypothesis comes from utrophin– dystrophin double-deficient mice, which show severe progressive muscle weakness, neuromuscular and myotendinous-junction abnormalities, and die prematurely.

Utrophin UpregulationUtrophin to treat dystrophin deficiency: Studies in mdx mice showed the feasibility of utrophin

upregulation to treat DMD. High expression of a truncated utrophin transgene at the

sarcolemma notably reduced dystrophic pathology and intracellular calcium homeostasis, and improved mechanical muscle performance.

Furthermore, adenoviral delivery of mini-utrophin restored the dystrophin–glycoprotein complex, reduced the number of centrally nucleated fibres and so rescued the dystrophic phenotypes of three animal models of DMD.

The expression of full-length utrophin mRNA in transgenic mdx mice produced even better results, despite expression levels that were at most 50% of the normal endogenous levels.

MYOBLAST TRANSPLANTATION

Is another dystrophin gene delivery strategy, also has problems that have prevented its use in the clinic: specifically immune rejection, limited cell spreading and poor survival of the myoblasts immediately post-transplantation.

Recently, similar cell-therapy strategies, which use stem cells that are derived from bone marrow, muscle or blood vessels, have had notable successes in dystrophic mouse models and DMD muscle.

Ex vivo gene therapyEx vivo gene therapy approaches using iPS cells

Induced pluripotent stem cells. A scheme for iPS cell-mediated ex vivo gene therapy approaches for DMD.

Skin fibroblasts or monocytes from peripheral blood are reprogrammed to iPS cells by transient expression of the Yamanaka factors.

The dystrophin mutation can then be repaired using genome engineering technologies. Such corrected iPS cells can be further differentiated into myoblasts to form myofibers.

Ex vivo gene therapyEither myoblasts or myofibers can be transplanted

to patients, but only for transient recovery, as myoblasts or myofibers will eventually die after cellular turnover.

An ideal approach would be to differentiate iPS cells into satellite cells, which are muscle stem cells, to gain long-term self-renewal and regeneration capacity in the myofibers.

Currently, ex vivo expansion of primary satellite and genome editing is challenging, but progress here could circumvent the use of iPS cells.

The future of DMD therapy

Steady progress in understanding the gene and its function has pointed to several innovative therapeutic strategies.

It now seems reasonable to expect that the next decade will see great advances in this field.

Considering its efficiency and relative simplicity, the antisense approach seems the next candidate (and probably the most promising so far) for clinical trials.

gene therapy for muscular dystrophy

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