Current therapeuticstrategies for Duchenne Muscular DystrophyIntroductionDuchenne MuscularDystrophy (DMD) is an X-linked, genetically inherited, debilitating, lifeshortening disease caused by a malfunction of the DMD gene.
The DMD gene is thelongest single gene in the human body which codes for dystrophin: a rod-shapedcytoplasmic protein, the protein is usually involved in joining actin fibres fromthe cytoskeleton and intracellular matrix contractile apparatus to theextracellular matrix1. Histological signs can be identified of DMDprior to any pathophysiological processes of the disease. In fact, foetaldiagnosis can be carried out as early as the third trimester of pregnancy. Thereare many exciting methods of potential treatment for DMD, the two most promisingat present could are those that involve the use of viral vectors or modifiednucleic acids. Pathophysiology of DMDDystrophin is part of an assembly of proteins responsiblefor strengthening cardiac and skeletal muscles and their protection from injuryduring muscular contraction and relaxation.
Specifically, dystrophin acts as ananchor protein, as stated and also can be found in nerve cells in the brainhowever their function is not affirmed however research suggests that they maybe associated with the usual function of synaptic transmission2.When Dystrophin is solubilised from its sarcolemmal fraction it becomes associatedwith glycoproteins and a large oligomeric complex, this forms the dystrophinassociated glycoprotein complex. This, in essence, forms the critical linkbetween the cytoskeleton and intracellular matrix contractile apparatus to theextracellular matrix in order for muscles to carry out persistent, undamagingmuscular contraction. Therefore, when the dystrophin associated glycoprotein complexmalfunctions, due to a dystrophin problem, the intuitive symptomatic featureswould be muscular related. The cause of the dystrophin protein beingnon-functional or partially functioning is due to a gene mutation in the DMDgene, most commonly a deletion, followed by point mutation and duplicationrespectively. DMD is characterised by, most commonly, an abnormal gait,frequent falls and difficulty climbing steps.
Less common symptomatic findingsinclude a reluctance to walk, poliomyelitis, delayed walking, walking on toesand excessive fatigue. Lumbar Lordosis is commonly affiliated with the waddlinggate found in DMD patients. Patients are often able to sustain a short periodof time stood on one leg however are unable to hop or turn in a usual form. Themost illustrative observational sign of DMD may be ‘the Gowers’ Manoeuvre’which shows a stereotypical sequence of postures shown in rising from a proneposition to an erect posture. However, Gower’s Manoeuvre is not pathognomonicas it is also present in other muscular disorders.
The six-minute walk test (6MWT)1,a recording of the distance that a patient can walk within six minutes, can beused as a method to monitor the progression of a disease such as DMD. DMDaffects around 1/5000 male births and those affected rarely live into theirfourth decade of life. Thus, there is clearly a desire for an effectivetreatment to halt this drastically life shortening life. Methodologies for muscular gene deliveryThere are a varietyof methods that involve the delivery of genes, specifically the DMD gene intothe human muscular system. One of the most exciting methods of gene insertionis using an Adeno Associated Virus (AAV), an asymptomatic, small virus whichcan be modified by inserting DNA to be used as a vector to deliver sections ofDNA into the human body. It is widely used due to its overall efficacy andsafety. The issue with DMD and using an AAV to deliver the DMD gene is therelative size of both subjects.
The DMD gene is a very large gene: 2.3megabases, the full-length cDNA is over 11kilobases whereas the AAV virus canusually capacitate just less than 5 kilobases. The question arises thereforehow do we effectively deliver the DMD gene, via AAV in attempt to treat DMD.
One method, seems to be to divide the full-length cDNA into three segments,each inserted into a vector (AAV) which all have unique recombination signalsfor directional recombination: Triple transplicing and reconstitution. Thismethod benefits due to the entire reconstitution of the full-length dystrophinbeing successfully expressed in mice. However, the efficiency of this methodmust be questioned due to low rates of successful vectoral reconstitution,however over time stronger and more unique recombination signals may lead togreater effectiveness rates. Equally as exciting is the report that there isthe potential that AAV can be engineered to express up to 15kb of gene, threetimes greater than that of a wild-type AAV genome, therefore furtheroptimisation of these methodologies may lead to expansion of the use of AAV ingene therapy3. The question asks however, is there a potential viralvector which either has a greater capacity for carrying DNA or is there a viralvector which responds greater to unique recombination signals, the latter Ifeel has been less investigated and therefore may lead to a potential newpathway of research. An alternativeapproach has also been made, using AAVs, to deliver a treatment of DMD. Thismethod involves using micro and mini-dystrophin, within an AAV vector in anattempt to address the symptoms of DMD.
Using mini-dystrophin is aimed at thereduction of the symptomatic effects of DMD to relate them more to thoseexperienced in mildly affected Becker’s Muscular Dystrophy (BMD). Micro-dystrophin,however, aims at the minimum requirement of the gene to function as normaldystrophin and that even in 50% correction would lead to a sufficient treatmentof cardiomyopathy in mdx mice4.In summary AAV can be used as a mediator to combine separate parts of the DMDgene or to carry truncated versions of the DMD gene in attempt to treat DMDhowever not eradicate the condition. Antisenseoligonucleotides (AONs), are nucleic acids, single stranded and chemicallymodified to target specific gene transcripts. Its small size is essential fordelivery and the chemical modifications that occur can affect: toxicity,affinity, solubility, stability and degradation resistance1.
Thereseem to be two main leading AONs: 2′-O-methyl phosphorothioate (2OMePs) andphosphorodiamidate morpholino oligomers (PMOs). The main difference between thetwo seems to be 2OMeP’s negative charge which leads to interactions with manyproteins, in particular the modulation of TLRs and leading to inflammatorysymptoms, renal failure and thrombocytopenia5. Therefore, it seems strangeto consider 2OMePs when PMOs induce little side effects whilst treatment showedpositive signs of revertant fibres in patient studies6. Therefore,there must be further research into which of PMOs and 2OMePs are more efficientor whether a cocktail of both AONs may be effective and if so whether there isa method of preventing the modulation of TLRs by 2OMePs. Relative Pre-Clinical Evidence In terms of AAV,beyond using mice with DMD, dogs become the next subject prior to clinicaltrials due to their greater complexity.
In terms of evidence the aim is toevaluate which serotype of AAV does not initiate a strong immune response,leading to a selectin of an AAV which will not be destroyed and potentiallythat will remain to promote Dystrophin creation for a longer period. AAV-1showed no protein expression when administered as a local limb muscle injectionand limb perfusion, in a young adult dog7. AAV-2 was carried out on normaldog muscle and led to an expression of cytotoxic T lymphocytes8.Most AAVs seem to either have a strong CTL production with no proteinexpression. However, AAV-8 had no CTL production and expression occurredhowever only for 2 minutes and was only on one specimen9. Therefore,it seems clear that to progress, more investigation needs to be taken onserotypes of AAV possibly specifically fine-tuning serotype AAV-8, as well astheir ability to carry micro- and mini-dystrophin and their potential abilityto function via triple or potentially double trans-splicing. In terms of usingAONs in pre-clinical research there is much compelling positive evidence. For example,in mdx52 mice using an injectioncontaining the 10 vPMO cocktail led to a 55% increase in the number ofdystrophin positive fibres produced.
Moreover, Western Blotting shows thatusing 10 vPMO can lead to full-length dystrophin expression10. Moreover,evidence that no adverse side effects were present in ten-fold doses innon-human primates indicates the safety of AONs and their suitability for usein fully clinical trials. Primates were euthanized in extremis using ketamine and an IV overdose of sodiumpentobartbital solution. The skin of the primates was then reflected from aventral midline incision and any abnormalities were identified. 59 differenttissue samples were examined. No effects related to treatment with AVI-4658were observed with respect to clinical observations11. This clearlyshows that the next step from these observations would be to carry out a humanclinical trial to monitor the efficacy, potency and the ability to remain inthe body by AONs and, seemingly more specifically, PMOs. Clinical EvidenceThere are currentlymany clinical trials in action and in different stages aiming at treating DMD,including trials concentrating on the AAV and AON methodology mentioned in thisessay.
However due to DMD research being a pioneering medical process manytrials have not had their results published or are in the further stages ofclinical trials. Using PMOs, specifically Eteplirsen, exhibited someimprovement in 7 of 19 patients. On average dystrophin fluorescence doubled inpatients on 2mg/kg/week and patients treated with 30 mg/kg/week had asignificant 22.
9% increase 24 weeks posttreatment. There was no such increasein the placebo grouping. After 48 weeks, there was a 51.7% increase. There wasno additional benefit for patients treated with 50mg/kg/week. However due tosome patients losing ambulation it was not a satisfactory end point of thetrain as determined by the FDA.
The main issue with PMOs seems to be the challengepresented by increased tissue-target uptake, this is due to the rapid clearanceof PMOs due to their neutral nature12. In terms of AAV, there are various research programmescurrently in session, mainly by Dr Jerry Mendell, including the administrationof rAAVrh74.MCK,µDystrophin13 intramuscularly toinvestigate whether this viral vector could be used as a potential treatmentfor DMD. Moreover, the trial of rAAV1.CMV.
huFollistatin34414will be used by Dr Mendell. Once these trials have been recorded, reviewed andpublished we may gain a greater insight on how far we have to go towards aneffective treatment of DMD. Discussion There are manydifferent viewpoints to be looked at when investigating the potential for acure/treatment of DMD. Challenges are presented due to the infancy of thetechniques used and also due to the systemic nature of the disease. There are,of course, other genetic methods that potentially may be able to treat DMD,such as CRISPR-Cas9, however it seems that the use of AONs, specifically PMOsin addition to the promising use of AAVs and their potential manipulation arethe most fore thinking and possibly closest to a true progression that mayresult in a beneficial alteration of the prognosis of DMD. References 1.Robinson-Hamm J, Gersbach C. Gene therapies that restore dystrophin expressionfor the treatment of Duchenne muscular dystrophy.
Human Genetics.2016;135(9):1029-1040.2. Reference G.DMD gene Internet.
Genetics Home Reference. 2017 cited 12 November 2017.Available from: https://ghr.nlm.nih.gov/gene/DMD#3.
Lostal W,Kodippili K, Yue Y, Duan D. Full-Length Dystrophin Reconstitution withAdeno-Associated Viral Vectors. Human Gene Therapy. 2014;25(6):552-562.
4.Athanasopoulos T, Graham I, Foster H, Dickson G. Recombinant adeno-associatedviral (rAAV) vectors as therapeutic tools for Duchenne muscular dystrophy(DMD). Gene Therapy.
2004;11(S1):S109-S121.5. Kole R,Krieg A. Exon skipping therapy for Duchenne muscular dystrophy. Advanced DrugDelivery Reviews. 2015;87:104-107.6. Fall A,Johnsen R, Honeyman K, Iversen P, Fletcher S, Wilton S.
Induction of revertantfibres in the mdx mouse using antisense oligonucleotides. Genetic Vaccines andTherapy. 2006;4(1):3.7. Vulin A,Barthélémy I, Goyenvalle A, Thibaud J, Beley C, Griffith G et al. MuscleFunction Recovery in Golden Retriever Muscular Dystrophy After AAV1-U7 ExonSkipping. Molecular Therapy. 2012;20(11):2120-2133.
8. Yuasa K,Yoshimura M, Urasawa N, Ohshima S, Howell J, Nakamura A et al. Injection of arecombinant AAV serotype 2 into canine skeletal muscles evokes strong immuneresponses against transgene products. Gene Therapy. 2007;14(17):1249-1260.9. Koo T, OkadaT, Athanasopoulos T, Foster H, Takeda S, Dickson G.
Long-term functionaladeno-associated virus-microdystrophin expression in the dystrophic CXMDj dog.The Journal of Gene Medicine. 2011;13(9):497-506.10.
Aoki Y,Yokota T, Nagata T, Nakamura A, Tanihata J, Saito T et al. Bodywide skipping ofexons 45-55 in dystrophic mdx52 mice by systemic antisense delivery.Proceedings of the National Academy of Sciences. 2012;109(34):13763-13768.11. Sazani P,Ness K, Weller D, Poage D, Palyada K, Shrewsbury S. Repeat-Dose ToxicologyEvaluation in Cynomolgus Monkeys of AVI-4658, a Phosphorodiamidate MorpholinoOligomer (PMO) Drug for the Treatment of Duchenne Muscular Dystrophy.International Journal of Toxicology.
2011;30(3):313-321.12. Lim K,Maruyama R, Yokota T. Eteplirsen in the treatment of Duchenne musculardystrophy.
Drug Design, Development and Therapy. 2017;Volume11:533-545.13. ClinicalIntramuscular Gene Transfer Trial of rAAVrh74.MCK.Micro-Dystrophin to PatientsWith Duchenne Muscular Dystrophy – Full Text View – ClinicalTrials.govInternet. Clinicaltrials.
gov. 2017 cited 12 November 2017. Available from:https://clinicaltrials.gov/ct2/show/NCT02376816?term=rAAVrh74.
MCK%2Cdystrophin=114. ClinicalIntramuscular Gene Transfer of rAAV1.CMV.huFollistatin344 Trial to PatientsWith Duchenne Muscular Dystrophy – Full Text View – ClinicalTrials.govInternet.
Clinicaltrials.gov. 2017 cited 12 November 2017. Available from:https://clinicaltrials.gov/ct2/show/NCT02354781?cond=rAAV1.CMV.huFollistatin344=1