In January 2017, Ionis Pharmaceuticals reported results of their phase 1/2 clinical trial of DMPKRx in subjects with DM1. While the field gained considerable insights into the compound, clinical endpoints and future clinical trial design, DMPKRx did not achieve sufficient exposure in skeletal muscle to have the desired effect on RNA splicing. An examination of the totality of data behind DMPKRx can yield further insights as Ionis develops the next generation of antisense oligonucleotide drug candidate for clinical trials in myotonic dystrophy (DM).

Preclinical Evidence Supported Development of Ionis’ Constrained Ethyl-modified Oligonucleotide for DM1

A strong collaborative team in academia and Ionis Pharmaceuticals has recently published their preclinical animal efficacy studies of ISIS 486178, a compound of a similar class to the DMPK antisense oligonucleotide used in the DM1 clinical trial, ISIS-DMPKRx.

The therapeutic candidate molecule, ISIS 486178, was selected after extensive optimization of both oligonucleotide sequence and backbone chemistry, with over 3,000 compounds screened for suppression of DMPK. The study evaluated a battery of molecular and functional endpoints in: (a) myotonic dystrophy type 1 (DM1) and control cell lines and (b) DMSXL mice dosed subcutaneously with the selected compound, ISIS 486178.

The candidate therapeutic produced a 70% reduction in expanded CUG repeat RNA and nuclear MBNL-RNA foci in mouse skeletal muscle and 30% reduction in cardiac muscle. DMSXL muscle histology, forelimb muscle grip strength and body weight were also improved, with no overt safety signals (endpoints: survival, liver enzymes, CPK, creatinine and genome-wide profiling) noted in either mice or cultured myotubes. Changes were not noted in brain DMPK RNA levels, a finding expected with systemic dosing of oligonucleotides. Prior studies of DM1 are supportive of muscle maturational defects as a component of the pathologic mechanism—treatment with ISIS 486178 largely restored the myofiber maturational profile in the soleus of DMSXL mice. DM1-related splicopathy is mild and variable in DMSXL, so drug effect on mis-splicing was not evaluated.

Preclinical Proof of Concept Achieved for Targeting Expanded DMPK RNA

Taken together, treatment of DMSXL mice with ISIS 486178 produced substantial and reproducible reduction in mutant DMPK transcripts, as well as phenotypic improvements. The constrained ethyl backbone chemistry used in ISIS 486178 exhibited differential exposure to two important DM1 targets, skeletal muscle (70% reduction in DMPK transcripts) versus heart (30%). Using their earlier generation oligonucleotide chemistry (2'-O-methoxyethyl modified or MOE), Ionis successfully partnered with Biogen to achieve sufficient CNS exposure after intrathecal delivery, ultimately leading to regulatory approval of Spinraza for all types of spinal muscular atrophy in late 2016. It appears that improving delivery of a DMPK-targeted antisense oligonucleotide is a viable path forward for DM1.

Next Steps

Data published by this investigative team provide a strong scientific rationale for targeting mutant DMPK with oligonucleotides operating by an RNase H mechanism. MDF has a BAC transgenic model under development at Jackson Laboratories that may be a better model for assessing efficacy in restoring splicing in the context of the DMPK locus, as well as assessing multi-system phenotypes. Finally, Ionis has publically announced an ongoing preclinical development program to obtain an antisense oligonucleotide with better exposure and intends to return to clinical trials in DM1.

Reference:

Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice.
Jauvin D, Chrétien J, Pandey SK, Martineau L, Revillod L, Bassez G, Lachon A, McLeod AR, Gourdon G, Wheeler TM, Thornton CA, Bennett CF, Puymirat J.
Mol Ther Nucleic Acids. 2017 Jun 16;7:465-474. doi: 10.1016/j.omtn.2017.05.007. Epub 2017 May 17.

Model organisms have yielded important insights into neuromuscular diseases. Findings from the relatively straightforward models now link unstable expansions of CTG and CTTG repeats to the phenotypes of myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2) respectively. Yet, it would be a mistake to assume that we understand all therapeutically relevant pathogenic or disease modifying mechanisms in DM. A particularly vexing issue has been how DM1 and DM2 are mediated by MBNL sequestration, but yield phenotypes of differing severity. New fly models may provide some insights.

Novel Models for DM1 and DM2

To address divergent aspects of pathology in DM1 and DM2, Dr. Rubén Artero and colleagues (University of Valencia) generated and evaluated novel Drosophila models expressing the respective repeats (250 CTG or 1,100 CCTG) in skeletal and cardiac muscle. Flies expressing 20 CTG or CCTG repeats were also generated and used as controls.

Similar, Severe Phenotypes Seen in DM1 and DM2 Fly Models

The investigators showed that the established molecular features of DM—formation of nuclear aggregates, MBNL depletion, RNA splicing defects and upregulation of autophagy genes (Atg4, Atg7, Atg8a, Atg9 and Atg12)—occurred in their DM1 and DM2 models. They establish that expanded CCUG repeat RNA has similar potential in vivo toxicity as does CUG repeat RNA. Both models had severe skeletal (50% reduction in fiber cross-sectional area) and cardiac muscle phenotypes, and reduced survival. Cardiac dysfunction included altered systolic and diastolic intervals, deficits in contractility (percentage (%) of fractional shortening) and arrhythmias; some cardiac measures showed higher severity in the DM2 model fly. 

Do Unknown Factors Mitigate Cardiac Disease in DM2?

While understanding that no model organism can actually be said to “have DM,” fly and mouse models have informed understanding and treatment of DM. In the DM2 fly model, the cardiac phenotype is more severe than is seen in DM2 patients. The investigators suggest that while both CUG and CCUG expanded repeat RNA have the potential to cause severe striated muscle phenotypes, there may be mechanisms beyond the well-established toxic RNA pathway that reduce the toxicity they observed in the fly in human DM2. These findings and models may have relevance for identification of genetic modifiers or as validation screens for small molecule drug development.

Reference:

Expanded CCUG Repeat RNA Expression in Drosophila Heart and Muscle Trigger Myotonic Dystrophy Type 1-like Phenotypes and Activate Autophagocytosis Genes.
Cerro-Herreros E, Chakraborty M, Pérez-Alonso M, Artero R, Llamusí B.
Sci Rep. 2017 Jun 6;7(1):2843. doi: 10.1038/s41598-017-02829-3.

DMPK CTG expansion length generally correlates with the severity of myotonic dystrophy type 1 (DM1), but is not fully prognostic of disease onset, course and severity. For congenital myotonic dystrophy (CDM), the apparent requirement for an epigenetic change upstream of the DMPK locus is apparently a co-requirement, along with a long CTG repeat. Moreover, the relationship between repeat expansion length and the cardiac phenotype in DM is a gap in our understanding of cardiac disease in DM1.

Multivariate Analysis of a Large Genetically Confirmed DM1 Cohort

Dr. Caroline Chong-Nguyen (Sorbonne Paris Cité University) and colleagues characterized the relationship between DMPK repeat expansion length and cardiac disease in a retrospective study of a cohort of 855 adult subjects from the DM1-Heart Registry. Subjects entered into the study had genetic analysis (Southern blot of peripheral blood) done at the time of their baseline cardiac investigations.

Genotyped patients were followed for a median of 11.5 years. The authors utilized a multivariate analysis that considered potential confounding factors, including age, sex, and diabetes mellitus.

Repeat Length is a Key Factor in Prognosis Even When Confounding Variables are Taken into Account

Survival of DM1 subjects was correlated with the quartile of CTG expansion size—37% mortality was reported in subjects with greater than 830 repeats. Across the range of repeat lengths studied, each 500 repeat increase was associated with 1.5-fold higher risk of death from all causes. Heart rate was higher and conduction system disease, left bundle branch block, and longer PR and QRS intervals were more prevalent in subjects with larger repeats. CTG length also associated with the presence of a permanently implanted pacemaker. Availability of extensive longitudinal data allowed the authors to report Kaplan–Meier estimates for survival, supraventricular arrhythmias, pacemaker implantation and sudden death.

This longitudinal study of a large cohort genotyped at the time of initial cardiac evaluation provides new insights into genotype-cardiac phenotype relationships in DM1. Overall, the authors showed that longer DMPK repeat expansions were correlated with the severity of cardiac involvement, including development of conduction defects, left ventricular dysfunction, supraventricular arrhythmias, the requirement for permanent pacing, sudden death and mortality. These findings support a more aggressive approach toward cardiac screening based on DMPK repeat length—the authors argue that care should be based on assessment of conduction system defects and other cardiac manifestations.

This peer-reviewed research article was accompanied by an editorial by Dr. Matthew Wheeler (Stanford University) in the same issue of the journal. This editorial is also referenced below.

References:

Association Between Mutation Size and Cardiac Involvement in Myotonic Dystrophy Type 1: An Analysis of the DM1-Heart Registry.
Chong-Nguyen C, Wahbi K, Algalarrondo V, Bécane HM, Radvanyi-Hoffman H, Arnaud P, Furling D, Lazarus A, Bassez G, Béhin A, Fayssoil A, Laforêt P, Stojkovic T, Eymard B, Duboc D.
Circ Cardiovasc Genet. 2017 Jun;10(3). pii: e001526. doi: 10.1161/CIRCGENETICS.116.001526.

Repeats and Survival in Myotonic Dystrophy Type 1.
Wheeler MT.
Circ Cardiovasc Genet. 2017 Jun;10(3). pii: e001783. doi: 10.1161/CIRCGENETICS.117.001783

MDF staff recently attended the 2017 annual meeting of the American Academy of Neurology, in Boston, MA. Here are highlights from that meeting.

Clinical and histopathological findings in myotonic muscular dystrophy type 2 (DM2): retrospective review of 49 DNA-confirmed cases.
Bhaskar Roy, Qian Wu, Charles Whitaker, and Kevin Felice.

A better understanding of the natural history of DM2 is essential to the design of interventional clinical trials. This poster reviewed clinical profiles of a cohort of 49 confirmed DM2 cases seen over 24-years at Beth Israel. Proximal lower limb weakness was the most predominant symptom, although weakness ranged from absent to severe. Myotonia, grip strength, and FVC also showed considerable variation. Approximately half of study subjects had cataracts.

Evaluation of postural control and falls in individuals with myotonic dystrophy type 1.
Katy Eichinger, Jill R. Quinn, and Shree Pandya.

Clinical trial endpoints that measure parameters meaningful to patients will be necessary for registration trials in myotonic dystrophy (DM1). This poster presented an assessment of postural control and self-reported falls in a cohort of 34 DM1 subjects, studied over a 12-week observational period. Postural sway measurements in DM1 subjects differed significantly from norms and showed good test/re-test reliability. None of the postural measures used were predictive of fall status, although this may be due to the small sample. Further evaluation of postural status may yield reliable, clinically meaningful clinical trial endpoints.

Identification of dysregulated musclin expression and elevated atrial natriuretic peptide levels in adult and congenital myotonic dystrophy.
Donald McCorquodale, Katie Mayne, Brith Otterud, Diane Dunn, Bob Weiss, and Nicholas Johnson.

Understanding tissue-level molecular changes in DM can guide biomarker development as well as identify novel therapeutic targets. This poster addressed two components of a pathway that mediates response to exercise. Musclin expression, an upstream regulator of atrial natriuretic peptide (ANP), increased in skeletal muscle of congenital myotonic dystrophy (CDM) and DM1, accompanied by increases in ANP clearance receptor (NPR3) and ANP. Disregulated musclin/ANP signaling may be linked to weakness and exercise intolerance in CDM and DM1.

Correlation between MRI cerebral white matter changes, muscle structure and/or muscle function in myotonic dystrophy type 1 (DM1).
Cheryl Smith, Peggy Nopoulos, Richard Shields, Dan Thedens, and Laurie Gutmann.

Understanding any linkage between CNS and skeletal muscle changes in DM1 may provide insights into putative biomarkers and clinical trial endpoints. This poster presented pilot data on potential CNS contributions to skeletal muscle structure and function in a DM1 cohort. Data show correlations between an MRI measure (global cerebral fraction anisotrophy—a measure of white matter abnormalities) and both MRI measures of lower limb muscle structure and a lower extremity tracking task (a measure of functional weight bearing movement). The authors concluded that these data suggest that CNS changes in DM1 play a role in neuromuscular functional deficits.

Borderline CNBP CCTG expansions in myotonic dystrophy type 2 in over 16,000 specimens analyzed in a clinical laboratory.
Elise Nedzweckas, Rebecca Moore, Marc Meservey, Tara McNamara, Nicholas Tiebout, Zhenyuan Wang, Sat Dev Batish, and Joseph Higgins.

The frequency of DM2 expansions in the pre-mutation range (CCTG repeat length of approximately 177-372) is unknown. This poster from Quest Diagnostics utilized PCR, PCR repeat-primed, and Southerns to determine CNBP CCTG expansion lengths in 16,253 samples. The frequency of ‘borderline’ repeats was 0.97%, a value larger than in previously published studies. The potential for repeats in this borderline range to expand to pathologic lengths is, as yet, unknown.

Genetic markers of myotonic dystrophy type 1 (DM1) and Duchenne muscular dystrophy (DMD) in human urine.
Layal Antoury, Ningyan Hu, Leonora Balaj, Xandra Breakfield, and Thurman Wheeler.

Availability of a non-invasive biomarker to track target engagement/modulation of candidate therapeutics would be valuable to any DM clinical trial, and elimination of muscle biopsies would be critical for trials in pediatric CDM subjects. The platform presentation reported analyses of exosomal RNA in blood and urine of DMD, BMD, and DM subjects. Serum showed no differences between DM1 and controls. Several splicing event alterations known to change in skeletal muscle were not detected in urine. But, at least 10 transcripts were differentially spliced in urine that followed patterns seen in skeletal muscle and thus showed potential as non-invasive biomarkers. The source of differentially spliced transcripts in urine was thought to be the kidney or other urinary tract cells.  The group is working to correlate the pattern of splicing events detected in urine with phenotypic changes in DM1 patients.

Receptor and post-receptor abnormalities contribute to insulin resistance in myotonic dystrophy type 1 and type 2 distal and proximal muscles.
Giovanni Meola, Laura Valentina Renna, Francesca Bose, Barbara Fossati, Elisa Brigozi, Michele Cavalli, and Rosana Cardani.

Metabolic dysfunction, including insulin resistance and increased risk of type 2 diabetes mellitus are characteristic of DM1 and DM2. While the insulin receptor (INSR) gene is known to be mis-spliced and links to the DM metabolic phenotype, other insulin signaling pathway components may be involved. This platform presentation presented data on insulin signaling pathway changes in DM1 and DM2 muscle biopsies. DM muscle biopsies showed increased fetal INSR isoform and altered expression and phosphorylation of selected proteins in the IR signaling pathway was seen in DM1 subjects. These effects were more pronounced in proximal versus distal muscles. The authors suggest that profiling of changes in INSR signaling pathways markers might emerge as a biomarker for clinical studies and trials in DM.

Increased EEG theta spectral power in polysomnography of myotonic dystrophy type 1 compared to matched controls.
Chad Ruoff, Joe Cheung, Jennifer Perez, Saranda Sakamuri, Emmanuel Mignot, John Day, and Jacinda Sampson.

Excessive daytime sleepiness and fatigue are hallmarks of DM—development of clinical endpoints to reliably evaluate these symptoms will help drive clinical trials. This poster presented data characterizing EEG spectra from nocturnal polysomnography in DM1 vs. controls. DM1 patients showed increases in wake after sleep onset and increased theta power in stage 2, stage 3, and all sleep stages combined when compared to control. EEG spectral power is being further evaluated as a putative biomarker.

There have been new discoveries in the way that congenital myotonic dystrophy (CDM) is inherited.

How is DM Inherited?

Myotonic dystrophy (DM) is inherited in what geneticists refer to as an autosomal dominant fashion. Let’s break that language down.  

Autosomal refers to the type of chromosome that carries the DM mutation—autosomes versus sex chromosomes. Humans have 23 pairs of chromosomes—pairs 1 through 22 have the same appearance in both males and females, and are referred to as autosomes; but pair 23 differs among the sexes (sex chromosomes), with two X chromosomes in females and one X and one Y in males. 

Autosomal then means that a mutation is carried on one of the chromosomal pairs 1 through 22. In the case of myotonic dystrophy (DM1), chromosome 19 carries the expanded CTG repeat mutation in the DMPK gene; for DM2, it’s chromosome 3 that carries the expanded CCTG repeat in the CNBP gene.

Dominant means that a mutation only has to be on one of the two members of a chromosomal pair to cause the disease. So, in DM1 (myotonic dystrophy type 1) it’s only necessary that the mutation in DMPK be on one member of the chromosome 19 pair, or one member of the chromosome 3 pair for DM2. Recessive disorders have to have the mutation on each member of a chromosomal pair and thus must be inherited from both parents.

Is Congenital DM the Same?

Autosomal dominant inheritance would typically mean that DM could be passed along by either parent. However, for congenital myotonic dystrophy (CDM), inheritance patterns are almost exclusively maternal. This maternal bias has been a mystery, since the “rules of genetics” would indicate that the likelihood of inheriting a DM mutation from either parent should be equal.

Drs. Karen Sermon (Vrije Universiteit Brussel) and Christopher E. Pearson (Hospital for Sick Children) and their colleagues explored the molecular basis for the maternal bias in the inheritance of CDM. They evaluated multiple generations of several families, including 20 individuals with CDM.

While the length of the CTG expansion was clearly greater in CDM than DM1, the investigators confirmed prior findings that the range of repeat lengths partially overlapped, suggesting that CTG repeat length was important, but not the only factor in determining whether someone had DM1 or the more severe CDM.

Genetic changes, such as the expanded CTG in DM1, can cause disease, but there are other changes in DNA that go beyond changes in the sequence of bases (A, T, C, G)—these changes also are heritable and referred to as epigenetic changes. To understand the basis of maternal inheritance bias in CDM, Sermon and Pearson looked for a pattern of epigenetic changes in the vicinity of the DMPK gene on chromosome 19.

The investigators identified specific epigenetic changes adjacent to DMPK in nearly every CDM patient studied, changes that were not observed in DM1 patients. CDM, therefore, appears to require both a long CTG repeat expansion and this epigenetic change. 

CDM and Maternal Bias

So, what causes the maternal bias in CDM inheritance? The investigators speculate that the maternal inheritance bias in CDM may be a consequence of failed survival of sperm that carry the epigenetic change in DMPK. Since sperm without the epigenetic change are at a survival disadvantage, the chances of paternal inheritance are considerably reduced. There are rare cases of paternal inheritance and these cases are sought by and under study by this research group to further advance understanding of CDM. For more information, access the research article.

The June issue of Current Opinion in Genetics and Development is focused on the topic "Molecular and Genetic Bases of Disease." Four outstanding review articles in the issue have direct relevance to myotonic dystrophy (DM) and are currently available online.

The first article (by Drs. Nan Zhang and Tetsuo Ashizawa) reviews the formation of RNA foci in microsatellite expansion disorders, how RNA binding proteins participate in toxic RNA gain of function, and how transcriptional and RNA processing/transport may ensue.

The second article (by Drs. Kevin Yum, Eric Wang, and Auinash Kalsotra) discusses mechanisms underlying repeat expansion, diagnostic approaches to determining repeat length, and how DM repeat length relates to disease onset, progression and severity.

The third article (by Drs. John Cleary and Laura Ranum) reviews basic mechanisms of repeat-associated non-ATG (RAN) translation and how RAN proteins may enter into the pathogenesis of microsatellite expansion disorders, including DM.

The fourth article (by Drs. Charles Thornton, Eric Wang and Ellie Carrell) focuses on the latest molecular strategies being used in the development of candidate therapies for DM, reviewing approaches targeted at transcriptional silencing, post-transcriptional silencing, inhibition of interactions between MBNL and toxic RNA, and pathways downstream of RNA toxicity.

Taken together, this is a compelling series of review articles, of benefit to basic, translational, and clinical scientists of all levels working on DM.

References:

RNA Toxicity and Foci Formation in Microsatellite Expansion Diseases
Zhang N, Ashizawa T.
Curr Opin Genet Dev. 2017 Feb 13;44:17-29. doi: 10.1016/j.gde.2017.01.005. [Epub ahead of print]

Myotonic Dystrophy: Disease Repeat Range, Penetrance, Age of Onset, and Relationship between Repeat Size and Phenotypes
Yum K, Wang ET, Kalsotra A.
Curr Opin Genet Dev. 2017 Feb 14;44:30-37. doi: 10.1016/j.gde.2017.01.007. [Epub ahead of print]

New Developments in RAN Translation: Insights from Multiple Diseases
Cleary JD, Ranum LP.
Curr Opin Genet Dev. 2017 Mar 30;44:125-134. doi: 10.1016/j.gde.2017.03.006. [Epub ahead of print]

Myotonic Dystrophy: Approach to Therapy
Thornton CA, Wang E, Carrell EM.
Curr Opin Genet Dev. 2017 Apr 1;44:135-140. doi: 10.1016/j.gde.2017.03.007. [Epub ahead of print]

Inheritance of congenital myotonic dystrophy (CDM) is almost exclusively maternal and, while typically associated with large CTG expansions, is not always genetically differentiated from myotonic dystrophy type 1 (DM1) by repeat tract length. Correlations between CDM/DM1 genotype and phenotype can be improved through evaluation of somatic expansions. Yet it is clear that factors other than germ line repeat length underlie the bias toward maternal inheritance and the heterogeneity of CDM.

The laboratories of Drs. Karen Sermon (Vrije Universiteit Brussel) and Chris Pearson (Hospital for Sick Children) recently collaborated on an epigenetic analysis of the DM1 genetic locus in a cohort of DM1 and CDM patients. Prior reports showed that the DM1 locus resides in a 3.5 kB CpG island with putative CTCF sites, suggesting an epigenetic mechanism for DM1 regulation and disease phenotypes that diverge from CTG length assessments. Earlier reports also established variability in methylation status at that locus in both DM1 patients and DM1 transgenic mice. Drs. Sermon and Pearson hypothesized that CTG expansion might alter CpG methylation status and that a consequent regulatory dysfunction contributes to the severity of the CDM phenotype.

Drs. Sermon and Pearson and team evaluated multiple generations of several families, including 20 individuals with CDM. Results showed nearly an absolute correlation between the methylation status upstream of the expanded CTG repeat and the occurrence of CDM (19/20 cases). By contrast, this pattern of methylation was rarely found among DM1 patients (2/59 cases). The authors suggest that CpG site methylation is an important contributing factor, with the development of CDM not being determined by CTG repeat length alone.

Analysis of human embryonic stem cells (hESC) and chorionic villus samples from the study cohort identified upstream CpG site methylation only in maternally-derived samples; paternal samples never showed methylation upstream of expanded DMPK alleles.

Generational increases in both methylation and CTG expansion length were seen in each CDM family studied. Yet since CTG repeat lengths overlapped in DM1 and CDM, while upstream methylation was almost exclusive to CDM, the authors concluded that methylation status is a stronger indicator of CDM than absolute repeat length. Moreover, they speculate that the maternal inheritance bias of CDM may be a consequence of a failed survival of spermatogonia carrying the pathogenic methylation upstream of DMPK. Importantly, while their data suggests that it is rare, the authors do not exclude paternal inheritance for CDM.

Reference:

CpG Methylation, a Parent-of-Origin Effect for Maternal-Biased Transmission of Congenital Myotonic Dystrophy.
Barbé L, Lanni S, López-Castel A, Franck S, Spits C, Keymolen K, Seneca S, Tomé S, Miron I, Letourneau J, Liang M, Choufani S, Weksberg R, Wilson MD, Sedlacek Z, Gagnon C, Musova Z, Chitayat D, Shannon P, Mathieu J, Sermon K, Pearson CE.
Am J Hum Genet. 2017 Mar 2;100(3):488-505. doi: 10.1016/j.ajhg.2017.01.033.

A potentially revolutionary technology may allow development of a drug for DM that can correct a patient’s DNA by selectively removing the expanded CTG and CCTG repeats in DM1 and DM2, respectively.

This new gene editing technology has emerged from the discovery of how bacteria protect themselves from invading viruses. There very likely will be a Nobel Prize awarded to scientists who discovered how this bacterial defense mechanism could be used to edit human gene defects. There certainly has been a rather public fight over the patent rights that are potentially worth billions of dollars among the research groups at the University of California, Berkeley and the Eli and Edythe L. Broad Institute of MIT and Harvard.

CRISPR is an acronym for short, repetitive DNA sequences that function to immunize bacteria from viruses. CRISPR DNA works together with a family of proteins, known as Cas proteins, which cut and thereby inactivate the invading virus’ DNA. Working together, CRISPR and Cas can recognize viral DNA as foreign and then inactivate it via the DNA-cutting Cas protein.

Researchers Jennifer Doudna (U.C. Berkeley) and Feng Zhang (Broad) have shown that the CRISPR/Cas system can be adapted to cut human DNA at highly specified locations to either remove existing genes or to insert new genes. The potential value for DM is that known mutation-containing DNA sequences, specifically the expanded repeats in DMPK or CNBP, could be cut to remove the disease-causing expanded repeats. Doudna and Zhang are likely to share a Nobel Prize for this discovery, while it appears that Zhang and the Broad Institute have won the patent rights battle.

Is gene editing using the CRISPR/Cas system going to be available soon for DM? The direct answer is, no. Several issues need to be resolved before any clinical application of gene editing is realized. The specificity and efficiency of gene editing will need to improve. Delivery of the gene editing reagents also must be optimized—these are large molecules that will have to be delivered by intravenous injections and must then gain access to cells throughout the body of DM patients in order to correct the multiple symptoms of the disease. Lastly, there is the issue of safety—studies need to show not only that the CRISPR/Cas system is designed to edit the DMPK or CNBP genes, but that it does not cause harm by editing unintended genes. MDF is working with researchers and biotechnology companies to help advance CRISPR/Cas gene editing for DM.

Gene editing is entering clinical trials this year for some types of cancer. The strategy is to edit the patient’s immune cells, outside of their body (thereby circumventing some barriers to the technology), and the cells that are put back into the body have gene edits so they attack the tumor. This human study is an important proof of concept, and a step that will be critical as we move toward the application of gene editing for DM.

Gene Editing

The basic science discovery of an adaptive immunity system that evolved in bacteria as a DNA-targeting viral defense mechanism was so revolutionary that early manuscripts from three independent labs were rejected by multiple journals. From that inauspicious beginning, CRISPR/Cas9 technology is rapidly advancing toward clinical trials in multiple disease indications. 

Even that renowned purveyor of scientific knowledge, The New Yorker, has touted the therapeutic potential of CRISPR/Cas9 gene editing (see "The Gene Hackers").

Gene editing is rapidly moving toward clinical trials, indeed ex vivo editing of T-cells in order to target tumors was approved by the National Institutes of Health’s (NIH) Recombinant DNA Advisory Committee (RAC) and trials are anticipated to start in early 2017. By design, the oncology clinical trial largely avoids many potential barriers to CRISPR/Cas9-based therapies—efficiency of gene editing, safety, delivery and ethics. It is not surprising that a disease where ex vivo gene editing is a plausible therapeutic strategy is the first to reach clinical trials.

Possibilities for DM

For myotonic dystrophy (DM), gene editing is an attractive, but currently theoretical strategy for directly addressing the primary genetic defect by excising pathogenic expanded CTG or CTTG repeats. Recognizing that expanded repeats are present in every cell, thereby requiring in vivo gene editing, the DM field must address all of the barriers noted above if clinical trials are to become a reality.

A recent publication from Dr. Bé Wieringa and colleagues takes an important step in assessing the feasibility of CRISPR/Cas9-mediated somatic gene editing for DM. In studies of myoblasts from normal subjects, DM1 patients, and immortalized mouse myoblasts (DM500), the research team explored ways of modulating efficacy of expanded repeat excision. They evaluated both unilateral (from one side of the repeat tract) and dual CRISPR cleavage strategies. 

The group’s findings show specificity in removal of normal and expanded CTG repeats from the DMPK locus. Their dual CRISPR editing approach resulted in unusually large deletions (kilobase size, encompassing the entire expanded repeat) with no adverse biologic consequences for gene expression, DMPK mRNA localization, MBNL distribution or myogenesis. Unilateral cleavage of an unstable genomic repeat was viewed as unadvised, since the ensuing recombination repair may produce unpredictable genomic changes.

The promise of gene editing lies in its objective of stopping downstream pathogenic mechanisms via correction of the primary DNA defect. The challenges lie in establishing safety (putative off-target editing), further optimization of editing efficacy/efficiency, and ensuring bioavailability of CRISPR/Cas9 reagents to tissues impacted by DM. These are not trivial barriers, but the latest findings by Dr. Wieringa and colleagues provide important in vitro proof of concept and thereby represent an important step.

Reference:

CRISPR/Cas9-Induced (CTG⋅CAG)n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing
van Agtmaal EL, André LM, Willemse M, Cumming SA, van Kessel ID, van den Broek WJ, Gourdon G, Furling D, Mouly V, Monckton DG, Wansink DG, Wieringa B.
Mol Ther. 2017 Jan 4;25(1):24-43. doi: 10.1016/j.ymthe. 2016.10.014.

Biomarkers are a major interest for myotonic dystrophy (DM), but understanding of their utility (Context of Use) in clinical trials can be elusive. The ‘flavors’ of biomarkers relate to the ways they are utilized: diagnostic, prognostic, predictive, pharmacodynamics (PD) and pharmacokinetic (PK). The right biomarkers are invaluable in selecting/stratifying patients, determining on-target activity, and dosing and assessing efficacy and safety of candidate therapies. Arriving at the “right” biomarkers to minimize uncertainty and aid decision-making is essential, but nontrivial, as experiences in Duchenne have so clearly shown.

A simplistic view is that a molecular endpoint identified in a laboratory study can be a panacea in accelerating drug approval. The reality is that, in moving beyond the discovery phase, a range of questions, from methodological to interpretive, must be answered before a biomarker has validity.

Given the high bar for regulatory qualification, biomarker studies ultimately must reside within collaborative networks that recognize that no one gets to the solution alone. A key barrier to overcome is that biomarker qualification is uncharted territory for most academic researchers.

Two recent initiatives, the BEST Resource (Biomarkers, EndpointS, and other Tools) and the Framework for Defining Evidentiary Criteria for Biomarker Qualification, are aimed at clarifying terminology and process with all stakeholders, and thereby accelerating qualification and use of biomarkers in therapy development programs.

The FDA-NIH Joint Leadership Council, dedicated to improving regulatory science, developed the BEST Resource. BEST initially focused on harmonizing the terminology of translational science and biomedical project development. The intent is to provide clarity and consistency in communications among all stakeholders. The BEST glossary is an invaluable resource, going well beyond biomarker and endpoint terminology to provide a wealth of examples and informational links.

The Framework for Defining Evidentiary Criteria for Biomarker Qualification, a partnership led by the Foundation for the NIH that includes NIH, FDA, PhRMA, the Critical Path Institute and pharmaceutical companies, provides “a general framework to assist the development of biomarkers for qualification, to improve upon the quality of submissions to the FDA and to clarify the evidentiary criteria needed to support the biomarker’s "Context Of Use" (pdf). The ready availability of these criteria increases transparency of the qualification process and thereby facilitates interactions between biomarker developers and FDA.

What opportunities exist to exploit these new tools in biomarker development for DM? In a recent DM Research News, MDF highlighted the potential for FDA biomarker qualification of a panel of splicing events identified with the Myotonic Dystrophy Clinical Research Network (DMCRN). The recent clarification of evidentiary standards will markedly aid this effort. In addition, the Wyck Foundation and MDF recently funded Dr. Thurman Wheeler (Massachusetts General Hospital) to explore miRNAs in serum and urine as DM1 biomarkers. While this is a discovery-phase project, it’s important that the new qualification guidance is taken into account even by studies at such an early stage.

A recent publication by Ms. Alessandra Perfetti, Dr. Fabio Martelli and colleagues (IRCCS Policlinico San Donato) piloted circulating miRNAs as putative biomarkers for DM1. Dysregulated miRNAs included miR-1, miR-27b, miR-133a/-133b, miR-140-3p, miR-206, miR-454 and miR-574. Elevated miRNAs correlated with impaired muscle strength and elevated MCK, and could readily distinguish DM1 (103 subjects) from controls (111). Some of the miRNAs identified in DM1 patient samples are non-specific in that they also are dysregulated in Duchenne (miR-1, mIR-206 and miR-133a/-133b), but that does not preclude their potential value as prognostic, predictive, PD or PK biomarkers in DM1. Before a biomarker can be qualified, more extensive studies must assess how this miRNA profile links to pathogenic or regenerative processes across multiple organ systems, and show if these miRNAs are suitable in tracking disease progression and/or drug efficacy.

To achieve qualified DM biomarkers, we all must speak the same “BEST” language and assimilate, rather than silo, lessons learned from each study. But most of all, the DM research community must adopt a highly collaborative culture (valuing community needs over individual publications), since validated, quantitative assays, well-powered and phenotypically rich data sets and inter-site validation are essential in navigating the pathway to effective drug development tools.

Reference:

BEST (Biomarkers, EndpointS, and other Tools) Resource
FDA-NIH Biomarker Working Group.

Framework for Defining Evidentiary Criteria for Biomarker Qualification: Final Version. Evidentiary Criteria Writing Group

Validation of Plasma MicroRNAs as Biomarkers for Myotonic Dystrophy Type 1.
Perfetti A, Greco S, Cardani R, Fossati B, Cuomo G, Valaperta R, Ambrogi F, Cortese A, Botta A, Mignarri A, Santoro M, Gaetano C, Costa E, Dotti MT, Silvestri G, Massa R, Meola G, Martelli F.
Sci Rep. 2016 Dec 1;6:38174. doi: 10.1038/srep38174.