Mutations in the MRPS28 gene encoding the small mitoribosomal subunit protein bS1m in a patient with intrauterine growth retardation, craniofacial dysmorphism and multisystemic involvement.

Mitochondria contain a dedicated translation system, which is responsible for the intramitochondrial synthesis of 13 mitochondrial DNA (mtDNA)-encoded polypeptides essential for the biogenesis of oxidative phosphorylation (OXPHOS) complexes I and III-V. Mutations in nuclear genes encoding factors involved in mitochondrial translation result in isolated or multiple OXPHOS deficiencies and mitochondrial disease. Here, we report the identification of disease-causing variants in the MRPS28 gene, encoding the small mitoribosomal subunit (mtSSU) protein bS1m in a patient with intrauterine growth retardation, craniofacial dysmorphism and developmental delay. Whole exome sequencing helped identify a seemingly homozygous missense variant NM_014018.2:c.356A>G, p.(Lys119Arg) which affected a highly conserved lysine residue. The variant was present in the mother in a heterozygous state, but not in the father who likely carried a large deletion spanning exon 2 and parts of introns 1 and 2 that could account for the apparent homozygosity of the patient. Polymerase chain reaction (PCR) amplification and Sanger sequencing of MRPS28 cDNA from patient fibroblasts revealed the presence of a truncated MRPS28 transcript, which lacked exon 2. Molecular and biochemical characterization of patient fibroblasts revealed a decrease in the abundance of the bS1m protein, decreased abundance of assembled mtSSU and inhibited mitochondrial translation. Consequently, OXPHOS biogenesis and cellular respiration were compromised in these cells. Expression of wild-type MRPS28 restored mitoribosomal assembly, mitochondrial translation and OXPHOS biogenesis, thereby demonstrating the deleterious nature of the identified MRPS28 variants. Thus, MRPS28 joins the increasing number of nuclear genes encoding mitoribosomal structural proteins linked to mitochondrial disease.

Variants in the MIPEP gene presenting with complex neurological phenotype without cardiomyopathy, impair OXPHOS protein maturation and lead to a reduced OXPHOS abundance in patient cells.

Most mitochondrial proteins are synthesized in the cytosol and targeted to mitochondria via N-terminal mitochondrial targeting signals (MTS) that are proteolytically removed upon import. Sometimes, MTS removal is followed by a cleavage of an octapeptide by the mitochondrial intermediate peptidase (MIP), encoded by the MIPEP gene. Previously, MIPEP variants were linked to four cases of multisystemic disorder presenting with cardiomyopathy, developmental delay, hypotonia and infantile lethality. We report here a patient carrying compound heterozygous MIPEP variants-one was not previously linked to mitochondrial disease-who did not have cardiomyopathy and who is alive at the age of 20 years. This patient had developmental delay, global hypotonia, mild optic neuropathy and mild ataxia. Functional characterization of patient fibroblasts and HEK293FT cells carrying MIPEP hypomorphic alleles demonstrated that deficient MIP activity was linked to impaired post-import processing of subunits from four of the five OXPHOS complexes and decreased abundance and activity of some of these complexes in human cells possibly underlying the development of mitochondrial disease. Thus, our work expands the genetic and clinical spectrum of MIPEP-linked disease and establishes MIP as an important regulator of OXPHOS biogenesis and function in human cells.

Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome.

The synthesis of all 13 mitochondrial DNA (mtDNA)-encoded protein subunits of the human oxidative phosphorylation (OXPHOS) system is carried out by mitochondrial ribosomes (mitoribosomes). Defects in the stability of mitoribosomal proteins or mitoribosome assembly impair mitochondrial protein translation, causing combined OXPHOS enzyme deficiency and clinical disease. Here we report four autosomal-recessive pathogenic mutations in the gene encoding the small mitoribosomal subunit protein, MRPS34, in six subjects from four unrelated families with Leigh syndrome and combined OXPHOS defects. Whole-exome sequencing was used to independently identify all variants. Two splice-site mutations were identified, including homozygous c.321+1G>T in a subject of Italian ancestry and homozygous c.322-10G>A in affected sibling pairs from two unrelated families of Puerto Rican descent. In addition, compound heterozygous MRPS34 mutations were identified in a proband of French ancestry; a missense (c.37G>A [p.Glu13Lys]) and a nonsense (c.94C>T [p.Gln32∗]) variant. We demonstrated that these mutations reduce MRPS34 protein levels and the synthesis of OXPHOS subunits encoded by mtDNA. Examination of the mitoribosome profile and quantitative proteomics showed that the mitochondrial translation defect was caused by destabilization of the small mitoribosomal subunit and impaired monosome assembly. Lentiviral-mediated expression of wild-type MRPS34 rescued the defect in mitochondrial translation observed in skin fibroblasts from affected subjects, confirming the pathogenicity of MRPS34 mutations. Our data establish that MRPS34 is required for normal function of the mitoribosome in humans and furthermore demonstrate the power of quantitative proteomic analysis to identify signatures of defects in specific cellular pathways in fibroblasts from subjects with inherited disease.

Direct delivery of Cas9 or base editor protein and guide RNA complex enables genome editing in the retina.

Genome editing by CRISPR-Cas holds promise for the treatment of retinal dystrophies. For therapeutic gene editing, transient delivery of CRISPR-Cas9 is preferable to viral delivery which leads to long-term expression with potential adverse consequences. Cas9 protein and its guide RNA, delivered as ribonucleoprotein (RNP) complexes, have been successfully delivered into the retinal pigment epithelium in vivo. However, the delivery into photoreceptors, the primary focus in retinal dystrophies, has not been achieved. Here, we investigate the feasibility of direct RNP delivery into photoreceptors and retinal pigment epithelium cells. We demonstrate that Cas9 or adenine-base editors complexed with guide RNA, can enter retinal cells without the addition of any carrier compounds. Once in the retinal cells, editing rates vary based on the efficacy of the guide RNA and the specific location edited within the genes. Cas9 RNP delivery at high concentrations, however, leads to outer retinal toxicity. This underscores the importance of improving delivery efficiency for potential therapeutic applications in the future.

New Editing Tools for Gene Therapy in Inherited Retinal Dystrophies.

Inherited retinal dystrophies (IRDs) are a heterogeneous group of diseases that affect more than 2 million people worldwide. Gene therapy (GT) has emerged as an exciting treatment modality with the potential to provide long-term benefit to patients. Today, gene addition is the most straightforward GT for autosomal recessive IRDs. However, there are three scenarios where this approach falls short. First, in autosomal dominant diseases caused by gain-of-function or dominant-negative mutations, the toxic mutated protein needs to be silenced. Second, a number of IRD genes exceed the limited carrying capacity of adeno-associated virus vectors. Third, there are still about 30% of patients with unknown mutations. In the first two contexts, precise editing tools, such as CRISPR-Cas9, base editors, or prime editors, are emerging as potential GT solutions for the treatment of IRDs. Here, we review gene editing tools based on CRISPR-Cas9 technology that have been used in vivo and the recent first-in-human application of CRISPR-Cas9 in an IRD.

Early and late stage gene therapy interventions for inherited retinal degenerations.

Inherited and age-related retinal degeneration is the hallmark of a large group of heterogeneous diseases and is the main cause of untreatable blindness today. Genetic factors play a major pathogenic role in retinal degenerations for both monogenic diseases (such as retinitis pigmentosa) and complex diseases with established genetic risk factors (such as age-related macular degeneration). Progress in genotyping techniques and back of the eye imaging are completing our understanding of these diseases and their manifestations in patient populations suffering from retinal degenerations. It is clear that whatever the genetic cause, the majority of vision loss in retinal diseases results from the loss of photoreceptor function. The timing and circumstances surrounding the loss of photoreceptor function determine the adequate therapeutic approach to use for each patient. Among such approaches, gene therapy is rapidly becoming a therapeutic reality applicable in the clinic. This massive move from laboratory work towards clinical application has been propelled by the advances in our understanding of disease genetics and mechanisms, gene delivery vectors, gene editing systems, and compensatory strategies for loss of photoreceptor function. Here, we provide an overview of existing modalities of retinal gene therapy and their relevance based on the needs of patient populations suffering from inherited retinal degenerations.