scholarly journals In vivo adenine base editing corrects newborn murine model of Hurler syndrome

2021 ◽  
Author(s):  
Jing Su ◽  
Xiu Jing ◽  
Kai qin She ◽  
Xiao mei Zhong ◽  
Qin yu Zhao ◽  
...  
Keyword(s):  
Murine Model ◽  
Human Condition ◽  
Type I ◽  
Newborn Mice ◽  
Base Editing ◽  
Minus Sign ◽  
Monogenic Diseases ◽  
Mps I ◽  
Adenine Base

Mucopolysaccharidosis type I (MPS I) is a severe disease caused by loss-of-function mutations variants in the α-L-iduronidase (IDUA) gene. In vivo genome editing represents a promising strategy to correct IDUA mutations, and has the potential to permanently restore IDUA function over the lifespan of the patients. Here, we used adenine base editing to directly convert A>G (TAG>TGG) in newborn murine model harboring Idua-W392X mutation, which recapitulates the human condition and is analogous to the highly prevalent human W402X mutation. We engineered a split-intein dual-adeno-associated virus (AAV) 9 in vivo adenine base editor to circumvent the package size limit of AAV vectors. Intravenous injection of AAV9-base editor system into MPS I newborn mice led to sustained enzyme expression sufficient for correction of metabolic disease (GAGs substrate accumulation) and prevention of neurobehavioral deficits. We observed a reversion of the W392X mutation in 22.46 plus-or-minus sign 6.74% of hepatocytes, 11.18 plus-or-minus sign 5.25% of heart and 0.34 plus-or-minus sign 0.12% of brain, along with decreased GAGs storage in peripheral organs (liver, spleen, lung and kidney). Collectively, these data showed the promise of a base editing approach to precisely correct a common genetic cause of MPS I in vivo and could be broadly applicable to the treatment of a wide array of monogenic diseases.

scholarly journals Sexual behaviour in a murine model of mucopolysaccharidosis type I (MPS I)

PLoS ONE ◽  
2019 ◽  
Vol 14 (12) ◽  
pp. e0220429 ◽  
Author(s):  
Ana Barbosa Mendes ◽  
Cinthia Castro do Nascimento ◽  
Vânia D’Almeida
Keyword(s):  
Sexual Behaviour ◽  
Murine Model ◽  
Type I ◽  
Mucopolysaccharidosis Type ◽  
Mucopolysaccharidosis Type I ◽  
Mps I

scholarly journals Characterization and validation of a preventative therapy for hypertrophic cardiomyopathy in a murine model of the disease

2020 ◽  
Vol 117 (37) ◽  
pp. 23113-23124
Author(s):  
Helena M. Viola ◽  
Ashay A. Shah ◽  
Victoria P. A. Johnstone ◽  
Henrietta Cserne Szappanos ◽  
Mark P. Hodson ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Hypertrophic Cardiomyopathy ◽  
Mitochondrial Membrane Potential ◽  
Metabolic Activity ◽  
Murine Model ◽  
Mitochondrial Membrane ◽  
Cardiac Myocyte ◽  
Human Condition ◽  
Preventative Therapy

Currently there is an unmet need for treatments that can prevent hypertrophic cardiomyopathy (HCM). Using a murine model we previously identified that HCM causing cardiac troponin I mutation Gly203Ser (cTnI-G203S) is associated with increased mitochondrial metabolic activity, consistent with the human condition. These alterations precede development of the cardiomyopathy. Here we examine the efficacy of in vivo treatment of cTnI-G203S mice with a peptide derived against the α-interaction domain of the cardiac L-type calcium channel (AID-TAT) on restoring mitochondrial metabolic activity, and preventing HCM. cTnI-G203S or age-matched wt mice were treated with active or inactive AID-TAT. Following treatment, targeted metabolomics was utilized to evaluate myocardial substrate metabolism. Cardiac myocyte mitochondrial metabolic activity was assessed as alterations in mitochondrial membrane potential and flavoprotein oxidation. Cardiac morphology and function were examined using echocardiography. Cardiac uptake was assessed using an in vivo multispectral imaging system. We identified alterations in six biochemical intermediates in cTnI-G203S hearts consistent with increased anaplerosis. We also reveal that AID-TAT treatment of precardiomyopathic cTnI-G203S mice, but not mice with established cardiomyopathy, restored cardiac myocyte mitochondrial membrane potential and flavoprotein oxidation, and prevented myocardial hypertrophy. Importantly, AID-TAT was rapidly targeted to the heart, and not retained by the liver or kidneys. Overall, we identify biomarkers of HCM resulting from the cTnI mutation Gly203Ser, and present a safe, preventative therapy for associated cardiomyopathy. Utilizing AID-TAT to modulate cardiac metabolic activity may be beneficial in preventing HCM in “at risk” patients with identified Gly203Ser gene mutations.

scholarly journals Adenine Base Editing in vivo with a Single Adeno-Associated Virus Vector

2021 ◽  
Author(s):  
Han Zhang ◽  
Nathan Bamidele ◽  
Pengpeng Liu ◽  
Ogooluwa Ojelabi ◽  
Xin D. Gao ◽  
...  
Keyword(s):  
Genetic Diseases ◽  
Cell Types ◽  
Vector System ◽  
Adeno Associated Virus ◽  
Base Editing ◽  
Guide Rna ◽  
Gene Therapies ◽  
Compact Size ◽  
Adenine Base

Base editors (BEs) have opened new avenues for the treatment of genetic diseases. However, advances in delivery approaches are needed to enable disease targeting of a broad range of tissues and cell types. Adeno-associated virus (AAV) vectors remain one of the most promising delivery vehicles for gene therapies. Currently, most BE/guide combinations and their promoters exceed the packaging limit (~5 kb) of AAVs. Dual-AAV delivery strategies often require high viral doses that impose safety concerns. In this study, we engineered an adenine base editor using a compact Cas9 from Neisseria meningitidis (Nme2Cas9). Compared to the well-characterized Streptococcus pyogenes Cas9-containing ABEs, Nme2-ABE possesses a distinct PAM (N4CC) and editing window, exhibits fewer off-target effects, and can efficiently install therapeutically relevant mutations in both human and mouse genomes. Importantly, we showed that in vivo delivery of Nme2-ABE and its guide RNA by a single-AAV vector can revert the disease mutation and phenotype in an adult mouse model of tyrosinemia. We anticipate that Nme2-ABE, by virtue of its compact size and broad targeting range, will enable a range of therapeutic applications with improved safety and efficacy due in part to packaging in a single-vector system.

scholarly journals Efficient precise in vivo base editing in adult dystrophic mice

2020 ◽  
Author(s):  
Li Xu ◽  
Chen Zhang ◽  
Haiwen Li ◽  
Peipei Wang ◽  
Yandi Gao ◽  
...  
Keyword(s):  
Rna Editing ◽  
Deep Sequencing ◽  
High Efficiency ◽  
Contractile Function ◽  
Functional Improvement ◽  
Base Editing ◽  
Monogenic Diseases ◽  
Editing Activity ◽  
Target Rna

ABSTRACTBackgroundRecent advances in the base editing technology have created an exciting opportunity to precisely correct disease-causing mutations. However, the large size of base editors and their inherited off-target activities pose challenges for in vivo base editing. Moreover, the requirement of a protospacer adjacent motif (PAM) sequence within a suitable window near the mutation site further limits the targeting feasibility. In this work, we rationally improved the adenine base editor (ABE) to overcome these challenges and demonstrated the exceptionally high efficiency to precisely edit the Duchenne muscular dystrophy (DMD) mutation in adult mice.MethodsWe employed a fluorescence reporter assay to assess the feasibility of ABE to correct the dystrophin mutation in mdx4cv mice. The intein protein trans-splicing (PTS) was used to split the oversized ABE into two halves for efficient packaging into adeno-associated virus 9 (AAV9). The ABE with broadened PAM recognition (ABE-NG) was rationally re-designed for improved off-target RNA editing activity and on-target DNA editing efficiency. The mdx4cv mice at the 5 weeks of age receiving intramuscular or intravenous injections of AAV9 carrying the improved ABE-NG were analyzed at 10 weeks or 10 months of age. The editing outcomes were analyzed by Sanger and deep sequencing of the amplicons, immunofluorescence staining, Western blot and contractile function measurements. The off-target activities, host immune response and long-term toxicity were analyzed by deep sequencing, ELISA and serological assays, respectively.ResultsWe showed efficient in vitro base correction of the dystrophin mutation carried in mdx4cv mice using ABE-NG. The super-fast intein-splits of ABE-NG enabled the expression of full-length ABE-NG and efficient AAV9 packaging. We rationally improved ABE-NG with eliminated off-target RNA editing activity and minimal PAM requirement, and packaged into AAV9 (AAV9-iNG). Intramuscular and intravenous administration of AAV9-iNG resulted in dystrophin restoration and functional improvement. At 10 months after AAV9-iNG treatment, a near complete rescue of dystrophin was measured in mdx4cv mouse hearts. The off-target activities remained low and no obvious toxicity was detected.ConclusionsThis study highlights the promise of permanent base editing using iABE-NG for the treatment of monogenic diseases, in particular, the genetic cardiomyopathies.

Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice

Science ◽  
2019 ◽  
pp. eaaw7166 ◽  
Author(s):  
Shuai Jin ◽  
Yuan Zong ◽  
Qiang Gao ◽  
Zixu Zhu ◽  
Yanpeng Wang ◽  
...  
Keyword(s):  
High Fidelity ◽  
Single Nucleotide Variants ◽  
Disease Treatment ◽  
Single Nucleotide ◽  
Genetic Changes ◽  
Base Editing ◽  
Guide Rna ◽  
Genome Wide ◽  
Adenine Base

Cytosine and adenine base editors (CBEs and ABEs) are promising new tools for achieving the precise genetic changes required for disease treatment and trait improvement. However, genome-wide and unbiased analyses of their off-target effects in vivo are still lacking. Our whole genome sequencing (WGS) analysis of rice plants treated with BE3, high-fidelity BE3 (HF1-BE3), or ABE revealed that BE3 and HF1-BE3, but not ABE, induce substantial genome-wide off-target mutations, which are mostly the C→T type of single nucleotide variants (SNVs) and appear to be enriched in genic regions. Notably, treatment of rice with BE3 or HF1-BE3 in the absence of single-guide RNA also results in the rise of genome-wide SNVs. Thus, the base editing unit of BE3 or HF1-BE3 needs to be optimized in order to attain high fidelity.

scholarly journals Computer simulations explain mutation-induced effects on the DNA editing by adenine base editors

2020 ◽  
Vol 6 (10) ◽  
pp. eaaz2309 ◽  
Author(s):  
Kartik L. Rallapalli ◽  
Alexis C. Komor ◽  
Francesco Paesani
Keyword(s):  
Conformational Changes ◽  
Single Mutation ◽  
Base Editing ◽  
Functional Roles ◽  
Dna Base ◽  
Dna Editing ◽  
Dynamics Simulations ◽  
Editing Enzyme ◽  
Adenine Base

Adenine base editors, which were developed by engineering a transfer RNA adenosine deaminase enzyme (TadA) into a DNA editing enzyme (TadA*), enable precise modification of A:T to G⋮C base pairs. Here, we use molecular dynamics simulations to uncover the structural and functional roles played by the initial mutations in the onset of the DNA editing activity by TadA*. Atomistic insights reveal that early mutations lead to intricate conformational changes in the structure of TadA*. In particular, the first mutation, Asp108Asn, induces an enhancement in the binding affinity of TadA to DNA. In silico and in vivo reversion analyses verify the importance of this single mutation in imparting functional promiscuity to TadA* and demonstrate that TadA* performs DNA base editing as a monomer rather than a dimer.

scholarly journals In vivo suppressor mutations correct a murine model of hereditary tyrosinemia type I

1999 ◽  
Vol 96 (21) ◽  
pp. 11928-11933 ◽  
Author(s):  
K. Manning ◽  
M. Al-Dhalimy ◽  
M. Finegold ◽  
M. Grompe
Keyword(s):  
Murine Model ◽  
Type I ◽  
Suppressor Mutations ◽  
Tyrosinemia Type I ◽  
Hereditary Tyrosinemia

scholarly journals In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels

2021 ◽  
Author(s):  
Tanja Rothgangl ◽  
Melissa K. Dennis ◽  
Paulo J. C. Lin ◽  
Rurika Oka ◽  
Dominik Witzigmann ◽  
...  
Keyword(s):  
Low Density Lipoprotein ◽  
Point Mutations ◽  
Density Lipoprotein ◽  
Negative Regulator ◽  
Base Editing ◽  
Guide Rna ◽  
Humoral Immune ◽  
Cholesterol Levels ◽  
Adenine Base

AbstractMost known pathogenic point mutations in humans are C•G to T•A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle–based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases.

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