in Rare Diseases
A MUTATION-INDEPENDENT APPROACH FOR MUSCULAR DYSTROPHY VIA UPREGULATION OF A MODIFIER GENE
Kemaladewi, D.U.*, Bassi, P.S.*, Erwood, S., Al-Basha, D., Gawlik, K.I., Lindsay, K., Hyatt, E., Kember, R., Place, K.M., Marks, R.M., Durbeej, M., Prescott, S.A., Ivakine, E.A., Cohn, R.D.
Nature. 2019. PMID: 31341277
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Neuromuscular disorders are often caused by heterogeneous mutations in large, structurally complex genes. Targeting compensatory modifier genes could be beneficial to improve disease phenotypes. Here we report a mutation-independent strategy to upregulate the expression of a disease-modifying gene associated with congenital muscular dystrophy type 1A (MDC1A) using the CRISPR activation system in mice.
MDC1A is caused by mutations in LAMA2 that lead to nonfunctional laminin-α2, which compromises the stability of muscle fibers and the myelination of peripheral nerves. Transgenic overexpression of Lama1, which encodes a structurally similar protein called laminin-α1, ameliorates muscle wasting and paralysis in mouse models of MDC1A, demonstrating its importance as a compensatory modifier of the disease1. However, postnatal upregulation of Lama1 is hampered by its large size, which exceeds the packaging capacity of vehicles that are clinically relevant for gene therapy. We modulate the expression of Lama1 in the dy2j/dy2j mouse model of MDC1A using an adeno-associated virus (AAV9) carrying a catalytically inactive Cas9 (dCas9), VP64 transactivators and single-guide RNAs that target the Lama1 promoter. When pre-symptomatic mice were treated, Lama1 was upregulated in skeletal muscles and peripheral nerves, which prevented muscle fibrosis and paralysis. In addition, we show that dystrophic features and disease progression were improved and reversed when the treatment was initiated in symptomatic dy2j/dy2j mice with apparent hindlimb paralysis and muscle fibrosis.
Collectively, our data demonstrate the feasibility and therapeutic benefit of CRISPR-dCas9-mediated upregulation of Lama1, which may enable mutation-independent treatment for all patients with MDC1A. This approach has broad applicability to a variety of disease-modifying genes and could serve as a therapeutic strategy for many inherited and acquired diseases.
DEVELOPMENT OF THERAPEUTIC GENOME ENGINEERING IN LAMA2-DEFICIENT CONGENITAL MUSCULAR DYSTROPHY
Kemaladewi, D.U., Cohn, R.D.
Emerging Topics in Life Sciences. 2019.
This review summarizes our preclinical efforts to develop CRISPR/Cas-based technologies for the treatment of LAMA2-deficient congenital muscular dystrophy (MDC1A), and discusses the challenges faced in the clinical translation of these strategies.
Take home messages:
- Fundamental studies such as the nature and biology of basement membranes, combined with the advancement of gene therapy and CRISPR/Cas9, contribute to considerable progress in the development of treatments for MDC1A.
- We were able to simultaneously remove a point mutation and create a functional donor splice site in the Lama2 gene, and demonstrate its therapeutic benefit in MDC1A mouse model.
- The application of CRISPR/dCas9 transcriptional upregulation of a disease modifier gene such as LAMA1 has the potential to be applied to all MDC1A patients, irrespective of their mutations.
- Similar to many other therapeutic modalities, the safety aspects concerning off-target effects, cytotoxicity and immune responses, as well as finding an optimum window of intervention and delivery methods require further studies. Overall, it should not minimize the enthusiasm to move CRISPR/Cas9-based therapies closer towards clinical application.
Cover Image: Colorful DNA & neuromuscular system.
It illustrates the mutations causing neuromuscular disorders and the wave of genetic-based therapies for these conditions. Each colorful shape represents individuals affected by rare diseases.
Design: Dwi Kemaladewi
INCREASED POLYAMINES AS PROTECTIVE DISEASE MODIFIERS IN CONGENITAL MUSCULAR DYSTROPHY
Kemaladewi, D.U., Benjamin, J.S., Hyatt, E., Ivakine, E.A., Cohn, R.D.
Human Molecular Genetics. 2018. PMID: 29566247
Most Mendelian disorders display extensive clinical heterogeneity that cannot be solely explained by primary genetic mutations. Instead, it is largely attributed to the presence of disease modifiers, which can exacerbate or lessen the severity of the disease. LAMA2-deficient congenital muscular dystrophy (LAMA2-CMD) is caused by mutations in the LAMA2 gene. Progressive muscle weakness is predominantly observed in the lower limbs in LAMA2-CMD patients, whereas upper limbs muscles are significantly less affected. However, very little is known about the molecular mechanism underlying differential pathophysiology between specific muscle groups.
Here, we demonstrate that the triceps muscles of the dy2j/dy2j mouse model of LAMA2-CMD demonstrate milder atrophy fibrosis, compared to the severely-affected tibialis anterior (TA) muscles, suggesting a protective mechanism in the upper limbs of these mice. Comparative gene expression analysis reveals that polyamine pathway components Amd1 and Smox contribute to this myopathic imbalanced, and targeted modulation of this pathway represents a novel therapeutic avenue for this devastating disease.
CORRECTION OF A SPLICING DEFECT IN A MOUSE MODEL OF CMD TYPE 1A USING A HDR-INDEPENDENT MECHANISM
Kemaladewi, D.U., Maino, E., Hyatt, E., Hou, H., Ding, M., Place, K.M., Zhu, X., Bassi, P.S., Baghestani, Z., Deshwar, A.G., Merico, D., Xiong, H.Y., Frey, B.J., Wilson, M.D., Ivakine, E.A., Cohn, R.D.
Nature Medicine. 2017. PMID: 28714989
Splice-site defects account for about 10% of pathogenic mutations that cause Mendelian diseases. Prevalence is higher in neuromuscular disorders (NMDs), owing to the unusually large size and multi-exonic nature of genes encoding muscle structural proteins. Therapeutic genome editing to correct disease-causing splice-site mutations has been accomplished only through the homology-directed repair (HDR) pathway, which is extremely inefficient in postmitotic tissues such as skeletal muscle.
Here, we describe a strategy using nonhomologous end-joining (NHEJ) to correct a pathogenic splice-site mutation causing congenital muscular dystrophy type 1A (MDC1A), which is characterized by severe muscle wasting and paralysis. Specifically, we correct a splice-site mutation that causes the exclusion of exon 2 from Lama2 mRNA and the truncation of Lama2 protein in the dy2J/dy2J mouse model of MDC1A. We showed that systemic delivery of AAV9 carrying CRISPR/Cas9 components simultaneously excise an intronic region containing the mutation and create a functional donor splice site through NHEJ. This strategy leads to the inclusion of exon 2 and restoration of full-length Lama2 protein, resulting in substantial improvement in muscle histopathology and function without signs of paralysis. Beyond MDC1A, this strategy is particularly attractive for the correction of underlying defects of genetic diseases caused by splice site mutations, especially those affecting tissues in which HDR is inefficient.
EXON SNIPPING IN DUCHENNE MUSCULAR DYSTROPHY
Kemaladewi, D.U., Cohn, R.D.
Trends in Molecular Medicine. 2016. PMID: 26856237
Duchenne muscular dystrophy (DMD) is a life-limiting neuromuscular disorder caused by mutations in the DMD gene encoding dystrophin. We discuss very recent studies that used CRISPR/Cas9 technology to ‘snip out’ mutated exons in DMD, restoring the reading frame of the gene. We also present cautionary aspects of translating this exciting technology into clinical practice.
SPELL CHECKING NATURE: VERSATILITY OF CRISPR/CAS9 FOR DEVELOPING TREATMENTS FOR INHERITED DISORDERS
Wojtal, D.*, Kemaladewi, D.U.*, Malam, Z., Abdullah, S., Wong, T.W.Y., Hyatt, E., Baghestani, Z., Pereira, S., Stavropoulos, J., Mouly, V., Mamchaoui, K.M., Muntoni, F., Voit, T., Gonorzky, H.D., Dowling, J.J., Wilson, M.D., Mendoza-Londono, R., Ivakine, E.A., Cohn, R.D.
* Equal contributions
American Journal of Human Genetics. 2016. PMID: 26686765
CRISPR/Cas9 has arisen as a frontrunner for efficient genome engineering, with potentially broad therapeutic implications. Here, we establish a pipeline that uses readily obtainable cells from affected individuals to investigate the therapeutic potential of CRISPR/Cas9 in a diverse set of genetic disorders.
We show that an adapted version of CRISPR/Cas9 increases the amount of utrophin, a known disease modifier in Duchenne muscular dystrophy (DMD). Furthermore, we demonstrate preferential elimination of the dominant-negative FGFR3 c.1138G>A allele in fibroblasts of an individual affected by achondroplasia. Using a previously undescribed approach involving single guide RNA, we successfully removed large genome rearrangement in primary cells of an individual with an X chromosome duplication including MECP2. Moreover, removal of a duplication of DMD exons 18–30 in myotubes of an individual affected by DMD produced full-length dystrophin. Our findings establish the far-reaching therapeutic utility of CRISPR/Cas9, which can be tailored to target numerous inherited disorders.