Considerations for Generating Humanized Mouse Models to Test Efficacy of Antisense Oligonucleotides

Vázquez-Domínguez, Irene; Garanto, Alejandro

doi:10.1007/978-1-0716-2010-6_18

Cited by 5 publications

(7 citation statements)

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“…To overcome this hurdle, a humanized rodent model where the target gene is replaced or partially replaced by the human counterpart could be used for the in vivo validation of the human NAT sequence. Deep phenotyping is required to assess whether the humanization recapitulates the disease phenotype or maintains the function of the gene [ 26 , 52 , 53 ]. Second to mouse models, zebrafish is also reported in our survey, likely due to the convenient genetic modification, well characterized development, high capacity, and rapid turnaround as a conventional experimental animal model [ 54 ].…”

Section: Discussionmentioning

confidence: 99%

Experimental Model Systems Used in the Preclinical Development of Nucleic Acid Therapeutics

Zhou

Arechavala‐Gomeza

Garanto

2023

Nucleic Acid Therapeutics

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Preclinical evaluation of nucleic acid therapeutics (NATs) in relevant experimental model systems is essential for NAT drug development. As part of COST Action “DARTER” (Delivery of Antisense RNA ThERapeutics), a network of researchers in the field of RNA therapeutics, we have conducted a survey on the experimental model systems routinely used by our members in preclinical NAT development. The questionnaire focused on both cellular and animal models. Our survey results suggest that skin fibroblast cultures derived from patients is the most commonly used cellular model, while induced pluripotent stem cell-derived models are also highly reported, highlighting the increasing potential of this technology. Splice-switching antisense oligonucleotide is the most frequently investigated RNA molecule, followed by small interfering RNA. Animal models are less prevalent but also widely used among groups in the network, with transgenic mouse models ranking the top. Concerning the research fields represented in our survey, the mostly studied disease area is neuromuscular disorders, followed by neurometabolic diseases and cancers. Brain, skeletal muscle, heart, and liver are the top four tissues of interest reported. We expect that this snapshot of the current preclinical models will facilitate decision making and the share of resources between academics and industry worldwide to facilitate the development of NATs.

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Section: Discussionmentioning

confidence: 99%

Experimental Model Systems Used in the Preclinical Development of Nucleic Acid Therapeutics

Zhou

Arechavala‐Gomeza

Garanto

2023

Nucleic Acid Therapeutics

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“…Personalized medicine holds a huge promise for severe genetic diseases ( Kim et al, 2019 ; Wang et al, 2020 ; Diener et al, 2022 ), and requires the appropriate animal models ( Kalmykov et al, 2018 ; Aartsma-Rus and van Putten, 2019 ; Polikarpova et al, 2022 ; Vázquez-Domínguez and Garanto, 2022 ). Sequence-specific RNA-based drugs, such as antisense oligonucleotides (ASO) and RNAi therapeutics ( Zhu et al, 2022 ; Zogg et al, 2022 ), are currently in development as a new generation of medicine to treat dominant neurological disorders such as Huntington’s disease, amyotrophic lateral sclerosis, spinocerebellar ataxia, and several others ( Zhao et al, 2017 ; Iannitti et al, 2018 ; Miniarikova et al, 2018 ; Southwell et al, 2018 ; Martier et al, 2019 ; Aimiuwu et al, 2020 ).…”

Section: Discussionmentioning

confidence: 99%

“…Sequence-specific RNA-based drugs, such as antisense oligonucleotides (ASO) and RNAi therapeutics ( Zhu et al, 2022 ; Zogg et al, 2022 ), are currently in development as a new generation of medicine to treat dominant neurological disorders such as Huntington’s disease, amyotrophic lateral sclerosis, spinocerebellar ataxia, and several others ( Zhao et al, 2017 ; Iannitti et al, 2018 ; Miniarikova et al, 2018 ; Southwell et al, 2018 ; Martier et al, 2019 ; Aimiuwu et al, 2020 ). Humanized mouse models are in demand to facilitate safety and efficacy studies of such innovative classes of drugs ( Nair et al, 2019 ; Zhu et al, 2019 ; Vázquez-Domínguez and Garanto, 2022 ). For instance, several humanized mouse models of Huntington’s disease are currently available for the preclinical evaluation of SNP-dependent ASO and RNAi-based gene therapy ( Miniarikova et al, 2018 ; Southwell et al, 2018 ).…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics

et al. 2023

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The development of personalized medicine for genetic diseases requires preclinical testing in the appropriate animal models. GNAO1 encephalopathy is a severe neurodevelopmental disorder caused by heterozygous de novo mutations in the GNAO1 gene. GNAO1 c.607 G>A is one of the most common pathogenic variants, and the mutant protein Gαo-G203R likely adversely affects neuronal signaling. As an innovative approach, sequence-specific RNA-based therapeutics such as antisense oligonucleotides or effectors of RNA interference are potentially applicable for selective suppression of the mutant GNAO1 transcript. While in vitro validation can be performed in patient-derived cells, a humanized mouse model to rule out the safety of RNA therapeutics is currently lacking. In the present work, we employed CRISPR/Cas9 technology to introduce a single-base substitution into exon 6 of the Gnao1 to replace the murine Gly203-coding triplet (GGG) with the codon used in the human gene (GGA). We verified that genome-editing did not interfere with the Gnao1 mRNA or Gαo protein synthesis and did not alter localization of the protein in the brain structures. The analysis of blastocysts revealed the off-target activity of the CRISPR/Cas9 complexes; however, no modifications of the predicted off-target sites were detected in the founder mouse. Histological staining confirmed the absence of abnormal changes in the brain of genome-edited mice. The created mouse model with the “humanized” fragment of the endogenous Gnao1 is suitable to rule out unintended targeting of the wild-type allele by RNA therapeutics directed at lowering GNAO1 c.607 G>A transcripts.

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“…Unfortunately, most of our sequences of interest are not completely conserved among different species. Therefore, the molecules designed to target a human sequence cannot be directly assessed in an animal model [ 55 ]. This means that for in vivo assessments, it is not usually possible to use exactly the same SSOs or U1snRNA sequences that are used for human cells.…”

Section: Splicing: How It Work and How It Can Be Modulatedmentioning

confidence: 99%

“…The alternative would be to generate humanized animal models, an approach that is both time- and resource-consuming and may contribute to a substantial increase in the drug development time while requiring additional funding. Furthermore, the generation of a humanized animal model for every mutation that needs to be targeted is neither feasible nor ethical and may not always recapitulate the human molecular and/or physiological phenotypes [ 54 , 55 ].…”

Section: Splicing: How It Work and How It Can Be Modulatedmentioning

confidence: 99%

Splicing Modulation as a Promising Therapeutic Strategy for Lysosomal Storage Disorders: The Mucopolysaccharidoses Example

Santos

Gonçalves

Matos

et al. 2022

Life

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Over recent decades, the many functions of RNA have become more evident. This molecule has been recognized not only as a carrier of genetic information, but also as a specific and essential regulator of gene expression. Different RNA species have been identified and novel and exciting roles have been unveiled. Quite remarkably, this explosion of novel RNA classes has increased the possibility for new therapeutic strategies that tap into RNA biology. Most of these drugs use nucleic acid analogues and take advantage of complementary base pairing to either mimic or antagonize the function of RNAs. Among the most successful RNA-based drugs are those that act at the pre-mRNA level to modulate or correct aberrant splicing patterns, which are caused by specific pathogenic variants. This approach is particularly tempting for monogenic disorders with associated splicing defects, especially when they are highly frequent among affected patients worldwide or within a specific population. With more than 600 mutations that cause disease affecting the pre-mRNA splicing process, we consider lysosomal storage diseases (LSDs) to be perfect candidates for this type of approach. Here, we introduce the overall rationale and general mechanisms of splicing modulation approaches and highlight the currently marketed formulations, which have been developed for non-lysosomal genetic disorders. We also extensively reviewed the existing preclinical studies on the potential of this sort of therapeutic strategy to recover aberrant splicing and increase enzyme activity in our diseases of interest: the LSDs. Special attention was paid to a particular subgroup of LSDs: the mucopolysaccharidoses (MPSs). By doing this, we hoped to unveil the unique therapeutic potential of the use of this sort of approach for LSDs as a whole.

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Considerations for Generating Humanized Mouse Models to Test Efficacy of Antisense Oligonucleotides

Cited by 5 publications

References 51 publications

Experimental Model Systems Used in the Preclinical Development of Nucleic Acid Therapeutics

Experimental Model Systems Used in the Preclinical Development of Nucleic Acid Therapeutics

CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics

Splicing Modulation as a Promising Therapeutic Strategy for Lysosomal Storage Disorders: The Mucopolysaccharidoses Example

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