Close

Therapeutic strategy for spinal muscular atrophy by combining gene supplementation and genome editing

Fumiyuki Hatanaka, Keiichiro Suzuki, Kensaku Shojima, Jingting Yu, Yuta Takahashi, Akihisa Sakamoto, Javier Prieto, Maxim Shokhirev, Concepcion Rodriguez Esteban, Estrella Nuñez-Delicado, Juan Carlos Izpisua Belmonte

Preprint posted on 6 April 2023 https://www.biorxiv.org/content/10.1101/2023.04.06.535786v1

Gene correction alone is not sufficient to treat spinal muscular atrophy (SMA). But combining gene editing with gene supplementation shows a more significant benefit in a mouse model of SMA.

Selected by Preethi Krishnaraj

Background

Spinal Muscular Atrophy (SMA) is a debilitating genetic disease characterized by muscle weakness and atrophy. This disease is caused by mutations in the SMN1 gene, which encodes the survival motor neuron 1 (SMN1) protein. Currently, the therapeutic strategies for SMA focus on prolonging life span and on improving the quality of the patients’ life. These include small molecule or gene supplementary therapies which aim to deliver a fully functional copy of SMN1. However, these strategies only have a beneficial effect for a limited amount of time and coming up with long-term therapies, such as the correction of the endogenous mutation, remains a significant challenge. In this preprint, the authors describe a novel gene therapy approach for treating spinal muscular atrophy (SMA) in a mouse model. They combined two existing therapies, gene supplementation and gene editing, into a single approach called Gene-DUET.

Homology-independent targeted integration

Genome editing using the CRISPR/Cas9 system exploits either homologous -directed repair (HDR), which is seen only in proliferating cells, or non-homologous end joining (NHEJ), which is seen both in proliferating and non-proliferating cells. To achieve CRISPR/Cas9 mediated knock-out, both HDR or NHEJ mechanisms can be used. However, achieving knock-ins – particularly in non-proliferating or differentiated cells – is more challenging as it requires HDR. In 2016, the lab of the preprint authors developed a CRISPR/Cas9- based genome editing technology which utilises NHEJ, known as homology-independent targeted integration (HITI). With this technique, the authors previously showed successful knock-in in non-proliferating cells1. The HITI donor plasmid includes the transgene which is flanked by Cas9 cleavage sites, alongside the sgRNA which specifies the target loci and the direction of insertion. The resulting Cas9 cleavage occurs both in the donor plasmid and the genomic target, thereby releasing the transgene from the donor which then gets incorporated at the site of the double strand break (DSB) in the genomic loci 1,2.

Key findings

AAV vectors for transducing non-dividing cells

Delivering therapeutic genes to the neurons in the spinal cord can be challenging. AAV vectors are often the first choice for safe delivery of therapeutic genes, and in the case of SMA, AAV-PHP.eB and AAV9 have been commonly used. To compare the efficacy of these two AAV vectors, the authors injected the respective AAV-GFP in the neonatal wildtype mouse and observed that AAV-PHP.eB was more efficient in transducing spinal motor neurons than AAV9.

HITI-mediated knock-in in spinal motor neurons

The authors then tested the in vivo efficacy of HITI in the spinal cord by delivering AAV-PHP.eB along with a guide RNA expression cassette into neonatal mice. The results showed GFP expression in the nucleus of motor neurons and liver, suggesting HITI-mediated knock-in is successful in spinal motor neurons.

Taken together, the findings described above show that AAV-PHP.eB is an efficient gene delivering carrier in the spinal cord and that HITI-mediated genome editing is successful in vivo in non-dividing, spinal motor neurons.

Synergistic effect of genome editing and gene supplementation

The authors used the HITI approach to correct the SMA mutation in the SMA mouse model. Although the HITI-treated mice showed improved phenotypes, the effect was insufficient for this treatment to be considered a long-term therapeutic strategy. It was observed that protein expression following gene editing was too late already for an effective rescue. Hence, in their next step, the authors developed the Gene-DUET method as part of which the AAV-PHP.eB vector was redesigned to include mSmn1 CDS for overexpression and the corrected mSmn1/SMN1 gene for genome editing (AAV-PHP.eB-SMN1-DUET). The authors then systematically delivered this vector into P0.5 SMA mice with or without Cas9, expressed by AAV-PHP.eB-Cas9 . Both the cDNA (without Cas9) and the DUET (with Cas9) restored molecular dysfunctions in the spinal cord, thereby improving the phenotype significantly. Furthermore, the authors reported that the Gene-DUET approach was more effective than cDNA supplementation only, as the effects were stable and long-lasting. This suggests it could be a promising approach for treating genetic diseases. 

Why I like this preprint

As someone who has had the chance to engage with SMA affected patients and hearing their therapeutic requirements, this preprint piqued my interest.

The Gene-DUET approach which was shown to be more effective than the traditional gene supplementation therapy might bring promising remedies for a wide range of neurological diseases in the future. Additionally, the finding that the AAV-PHP.eB can efficiently transduce spinal neurons can have important implications for therapies for neurological disease. Overall, these findings provide the basis for further research and development for SMA- and related diseases.

Questions to the authors

  1. What safety concerns could the Gene-DUET system pose if it is to be implemented for clinical trials?
  2. What are the limitations of the HITI technology in terms of efficiency, specificity and toxicity?
  3. What further research is needed to fully explore the potential of the HITI strategy for treating genetic diseases?

References

  1. Suzuki, K. et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540, 144–149 (2016).
  2. Suzuki, K. & Izpisua Belmonte, J. C. In vivo genome editing via the HITI method as a tool for gene therapy. J. Hum. Genet. 63, 157–164 (2018).

Tags: crispr, gene therapy, gene-duet, neurological disease, spinal muscular atrophy

Posted on: 3 May 2023 , updated on: 12 May 2023

doi: https://doi.org/10.1242/prelights.34510

Read preprint (No Ratings Yet)

Have your say

Your email address will not be published.

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

Also in the developmental biology category:

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.

 



List by Helen Zenner

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.

 



List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.

 



List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

2nd Conference of the Visegrád Group Society for Developmental Biology

Preprints from the 2nd Conference of the Visegrád Group Society for Developmental Biology (2-5 September, 2021, Szeged, Hungary)

 



List by Nándor Lipták

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

EMBL Conference: From functional genomics to systems biology

Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020

 



List by Jesus Victorino

Single Cell Biology 2020

A list of preprints mentioned at the Wellcome Genome Campus Single Cell Biology 2020 meeting.

 



List by Alex Eve

Society for Developmental Biology 79th Annual Meeting

Preprints at SDB 2020

 



List by Irepan Salvador-Martinez, Martin Estermann

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EDBC Alicante 2019

Preprints presented at the European Developmental Biology Congress (EDBC) in Alicante, October 23-26 2019.

 



List by Sergio Menchero et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

SDB 78th Annual Meeting 2019

A curation of the preprints presented at the SDB meeting in Boston, July 26-30 2019. The preList will be updated throughout the duration of the meeting.

 



List by Alex Eve

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Young Embryologist Network Conference 2019

Preprints presented at the Young Embryologist Network 2019 conference, 13 May, The Francis Crick Institute, London

 



List by Alex Eve

Pattern formation during development

The aim of this preList is to integrate results about the mechanisms that govern patterning during development, from genes implicated in the processes to theoritical models of pattern formation in nature.

 



List by Alexa Sadier

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

Zebrafish immunology

A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.

 



List by Shikha Nayar

Also in the genetics category:

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.

 



List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.

 



List by Sergio Menchero et al.

Semmelweis Symposium 2022: 40th anniversary of international medical education at Semmelweis University

This preList contains preprints discussed during the 'Semmelweis Symposium 2022' (7-9 November), organised around the 40th anniversary of international medical education at Semmelweis University covering a wide range of topics.

 



List by Nándor Lipták

20th “Genetics Workshops in Hungary”, Szeged (25th, September)

In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link: http://group.szbk.u-szeged.hu/minikonf/archive/prg2021.pdf

 



List by Nándor Lipták

2nd Conference of the Visegrád Group Society for Developmental Biology

Preprints from the 2nd Conference of the Visegrád Group Society for Developmental Biology (2-5 September, 2021, Szeged, Hungary)

 



List by Nándor Lipták

EMBL Conference: From functional genomics to systems biology

Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020

 



List by Jesus Victorino

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill
Close