bims-algemi Biomed News
on Allotopic expression and gene therapy for mitochondrial disease
Issue of 2020‒02‒23
twenty papers selected by
Atif Towheed
Columbia University Irving Medical Center

  1. EMBO Rep. 2020 Feb 19. e49612
    Nissanka N, Moraes CT.
      Mitochondrial DNA (mtDNA) encodes a subset of the genes which are responsible for oxidative phosphorylation. Pathogenic mutations in the human mtDNA are often heteroplasmic, where wild-type mtDNA species co-exist with the pathogenic mtDNA and a bioenergetic defect is only seen when the pathogenic mtDNA percentage surpasses a threshold for biochemical manifestations. mtDNA segregation during germline development can explain some of the extreme variation in heteroplasmy from one generation to the next. Patients with high heteroplasmy for deleterious mtDNA species will likely suffer from bona-fide mitochondrial diseases, which currently have no cure. Shifting mtDNA heteroplasmy toward the wild-type mtDNA species could provide a therapeutic option to patients. Mitochondrially targeted engineered nucleases, such as mitoTALENs and mitoZFNs, have been used in vitro in human cells harboring pathogenic patient-derived mtDNA mutations and more recently in vivo in a mouse model of a pathogenic mtDNA point mutation. These gene therapy tools for shifting mtDNA heteroplasmy can also be used in conjunction with other therapies aimed at eliminating and/or preventing the transfer of pathogenic mtDNA from mother to child.
    Keywords:  gene editing; heteroplasmy; mitochondrial DNA
  2. J Diabetes Res. 2020 ;2020 2057187
    Li K, Wu L, Liu J, Lin W, Qi Q, Zhao T.
      We report here the clinical, genetic, and molecular characteristics of type 2 diabetes in a Chinese family. There are differences in the severity and age of onset in diabetes among these families. By molecular analysis of the complete mitochondrial genome in this family, we identified the homoplasmic m.15897G>A mutation underwent sequence analysis of whole mitochondrial DNA genome, which localized at conventional position ten of tRNAThr, and distinct sets of mtDNA polymorphisms belonging to haplogroup D4b1. This mutation has been implicated to be important for tRNA identity and stability. Using cybrid cell models, the decreased efficiency of mitochondrial tRNAThr levels caused by the m.15897G>A mutation results in respiratory deficiency, protein synthesis and assembly, mitochondrial ATP synthesis, and mitochondrial membrane potential. These mitochondrial dysfunctions caused an increase in the production of reactive oxygen species in the mutant cell lines. These data provide a direct evidence that a novel tRNA mutation was associated with T2DM. Thus, our findings provide a new insight into the understanding of pathophysiology of maternally inherited diabetes.
  3. JBRA Assist Reprod. 2020 Feb 19.
    Farnezi HCM, Goulart ACX, Santos AD, Ramos MG, Penna MLF.
      The mitochondria are intracellular organelles, and just like the cell nucleus they have their own genome. They are extremely important for normal body functioning and are responsible for ATP production - the main energy source for the cell. Mitochondrial diseases are associated with mutations in mitochondrial DNA and are inherited exclusively from the mother. They can affect organs that depend on energy metabolism, such as skeletal muscles, the cardiac system, the central nervous system, the endocrine system, the retina and liver, causing various incurable diseases. Mitochondrial replacement techniques provide women with mitochondrial defects a chance to have normal biological children. The goal of such treatment is to reconstruct functional oocytes and zygotes, in order to avoid the inheritance of mutated genes; for this the nuclear genome is withdrawn from an oocyte or zygotes, which carries mitochondrial mutations, and is implanted in a normal anucleated cell donor. Currently, the options of a couple to prevent the transmission of mitochondrial diseases are limited, and mitochondrial donation techniques provide women with mitochondrial defects a chance to have normal children. The nuclear genome can be transferred from oocytes or zygotes using techniques such as pronuclear transfer, spindle transfer, polar body transfer and germinal vesicle transfer. This study presents a review of developed mitochondrial substitution techniques, and its ability to prevent hereditary diseases.
    Keywords:  mitochondrial; mitochondrial donation; mitochondrial mutations; mitochondrial replacement; mtDNA; reproductive technology
  4. Biol Chem. 2020 Feb 01. pii: /j/bchm.just-accepted/hsz-2020-0114/hsz-2020-0114.xml. [Epub ahead of print]
    Edwards R, Gerlich S, Tokatlidis K.
      The mitochondrial intermembrane space (IMS) houses a large spectrum of proteins with distinct and critical functions. Protein import into this mitochondrial sub-compartment is underpinned by an intriguing variety of pathways, many of which are still poorly understood. The constricted volume of the IMS and the topological segregation by the inner membrane cristae into a bulk area surrounded by the boundary inner membrane and the lumen within the cristae is an important factor that adds to the complexity of the protein import, folding and assembly processes. We discuss the main import pathways into the IMS, but also how IMS proteins are degraded or even retro-translocated to the cytosol in an integrated network of interactions that is necessary to maintain a healthy balance of IMS proteins under physiological and cellular stress conditions. We conclude this review by highlighting new and exciting perspectives in this area with a view to develop a better understanding of yet unknown, likely unconventional import pathways, how presequence-less proteins can be targeted and the basis for dual localisation in the IMS and the cytosol. Such knowledge is critical to understand the dynamic changes of the IMS proteome in response to stress, and particularly important for maintaining optimal mitochondrial fitness.
    Keywords:  intermembrane space; mitochondria; oxidative folding; protein folding; protein import
  5. Hear Res. 2020 Feb 10. pii: S0378-5955(19)30474-5. [Epub ahead of print] 107912
    Akil O.
      Adeno-associated virus (AAV)-mediated gene therapy has evolved from the bench to the bedside, and is now considered the therapy of choice for certain inherited diseases. AAVs are attractive vectors for several reasons: they are nonpathogenic, result in long-term transgene expression, have a low immunogenic profile, and the various AAV serotypes and variants display broad but distinct tropisms allowing the targeting of specific cell types. However, one of the greatest limitations of AAVs is the limited genome-packaging capacity of ∼4.7 kb. Given that numerous diseases are caused by mutations in genes with coding sequences exceeding this capacity, packaging into a single AAV capsid is currently unfeasible for larger genes. Taking advantage of the AAV genome's ability to concatemerize, multiple strategies have been explored to overcome the size limit of AAV vectors. One strategy is to split large transgenes into two or three parts, generating dual or triple AAV vectors. Coinfection of a cell with these two or three AAVs will then, through a variety of mechanisms, result in the transcription of an assembled mRNA that could not be encoded by a single AAV vector. This review: 1) documents AAV dual and triple vector strategies currently employed in a variety of tissues, and highlights the advantages and disadvantages of each method; 2) describes the first successful studies using the dual vector approach to restore hearing and prevent deafness in a mouse model of non-syndromic deafness due to absence of the otoferlin protein function, and the implications of these findings for the future of gene therapy in the human inner ear; and 3) highlights additional different deafness genes that could be potential future targets for gene therapy using the dual vector approach.
    Keywords:  Adeno-associated virus (AAV) vectors; Cochlea; Dual and triple AAV vector approach; Gene therapy; Hearing loss; Otoferlin
  6. Front Neurosci. 2019 ;13 1444
    Llanos-González E, Henares-Chavarino ÁA, Pedrero-Prieto CM, García-Carpintero S, Frontiñán-Rubio J, Sancho-Bielsa FJ, Alcain FJ, Peinado JR, Rabanal-Ruíz Y, Durán-Prado M.
      Although the basis of Alzheimer's disease (AD) etiology remains unknown, oxidative stress (OS) has been recognized as a prodromal factor associated to its progression. OS refers to an imbalance between oxidant and antioxidant systems, which usually consist in an overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) which overwhelms the intrinsic antioxidant defenses. Due to this increased production of ROS and RNS, several biological functions such as glucose metabolism or synaptic activity are impaired. In AD, growing evidence links the ROS-mediated damages with molecular targets including mitochondrial dynamics and function, protein quality control system, and autophagic pathways, affecting the proteostasis balance. In this scenario, OS should be considered as not only a major feature in the pathophysiology of AD but also a potential target to combat the progression of the disease. In this review, we will discuss the role of OS in mitochondrial dysfunction, protein quality control systems, and autophagy associated to AD and suggest innovative therapeutic strategies based on a better understanding of the role of OS and proteostasis.
    Keywords:  Alzheimer’s disease; autophagy; endoplasmic reticulum stress; oxidative stress; proteasomal degradation; unfolded protein response
  7. Gene Ther. 2020 Feb 17.
    Watano R, Ohmori T, Hishikawa S, Sakata A, Mizukami H.
      Adeno-associated virus (AAV) vectors can transduce hepatocytes efficiently in vivo in various animal species, including humans. Few reports, however, have examined the utility of pigs in gene therapy. Pigs are potentially useful in preclinical studies because of their anatomical and physiological similarity to humans. Here, we evaluated the utility of microminipigs for liver-targeted gene therapy. These pigs were intravenously inoculated with an AAV8 vector encoding the luciferase gene, and gene expression was assessed by an in vivo imaging system. Robust transgene expression was observed almost exclusively in the liver, even though the pig showed a low-titer of neutralizing antibody (NAb) against the AAV8 capsid. We assessed the action of NAbs against AAV, which interfere with AAV vector-mediated gene transfer by intravascular delivery. When a standard dose of vector was administered intravenously, transgene expression was observed in both NAb-negative and low-titer (14×)-positive subjects, whereas gene expression was not observed in animals with higher titers (56×). These results are compatible with our previous observations using nonhuman primates, indicating that pigs are useful in gene therapy experiments, and that the role of low-titer NAb in intravenous administration of the AAV vector shows similarities across species.
  8. Curr Opin Obstet Gynecol. 2020 Feb 14.
    Cozzolino M, Seli E.
      PURPOSE OF REVIEW: To provide an overview of mitochondrial functional alterations in women with polycystic ovary syndrome (PCOS).RECENT FINDINGS: Although numerous studies have focused on PCOS, the pathophysiological mechanisms that cause this common disease remain unclear. Mitochondria play a central role in energy production, and mitochondrial dysfunction may underlie several abnormalities observed in women with PCOS. Recent studies associated mtDNA mutations and low mtDNA copy number with PCOS, and set out to characterize the potential protective role of mitochondrial and endoplasmic reticulum unfolded protein responses (UPR and UPR).
    SUMMARY: Mitochondrial dysfunction likely plays a role in the pathogenesis of PCOS by increasing reactive oxygen (ROS) and oxidative stress. This occurs in a metabolic milieu often affected by insulin resistance, which is a common finding in women with PCOS, especially in those who are overweight or obese. Mutations in mtDNA and low mtDNA copy number are found in these patients and may have potential as diagnostic modalities for specific PCOS phenotypes. More recently, UPR and UPR are being investigated as potential cellular rescue mechanisms in PCOS, the failure of which may lead to apoptosis, and contribute to decreased reproductive potential.
  9. Biochim Biophys Acta Mol Basis Dis. 2020 Feb 13. pii: S0925-4439(20)30071-5. [Epub ahead of print] 165726
    Villanueva-Paz M, Povea-Cabello S, Villalón-García I, Álvarez-Córdoba M, Suárez-Rivero JM, Talaverón-Rey M, Jackson S, Falcón-Moya R, Rodríguez-Moreno A, Sánchez-Alcázar JA.
      Mitochondrial diseases are considered rare genetic disorders characterized by defects in oxidative phosphorylation (OXPHOS). They can be provoked by mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). MERRF (Myoclonic Epilepsy with Ragged-Red Fibers) syndrome is one of the most frequent mitochondrial diseases, principally caused by the m.8344A>G mutation in mtDNA, which affects the translation of all mtDNA-encoded proteins and therefore impairs mitochondrial function. In the present work, we evaluated autophagy and mitophagy flux in transmitochondrial cybrids and fibroblasts derived from a MERRF patient, reporting that Parkin-mediated mitophagy is increased in MERRF cell cultures. Our results suggest that supplementation with coenzyme Q10 (CoQ), a component of the electron transport chain (ETC) and lipid antioxidant, prevents Parkin translocation to the mitochondria. In addition, CoQ acts as an enhancer of autophagy and mitophagy flux, which partially improves cell pathophysiology. The significance of Parkin-mediated mitophagy in cell survival was evaluated by silencing the expression of Parkin in MERRF cybrids. Our results show that mitophagy acts as a cell survival mechanism in mutant cells. To confirm these results in one of the main affected cell types in MERRF syndrome, mutant induced neurons (iNs) were generated by direct reprogramming of patients-derived skin fibroblasts. The treatment of MERRF iNs with Guttaquinon CoQ10 (GuttaQ), a water-soluble derivative of CoQ, revealed a significant improvement in cell bioenergetics. These results indicate that iNs, along with fibroblasts and cybrids, can be utilized as reliable cellular models to shed light on disease pathomechanisms as well as for drug screening.
    Keywords:  Autophagy; Coenzyme Q(10); Mitochondria; Mitochondrial diseases; Mitophagy
  10. Curr Gene Ther. 2020 Feb 17.
    Taheri F, Taghizadeh E, Pour MJR, Rostami D, Renani PG, Rastgar-Moghadam A, Hayat SMG.
      The limb-girdle muscular dystrophies (LGMD) are genetically heterogeneous disorders, responsible for muscle wasting and severe form of dystrophies. Despite the critical developments in the insight and information of pathomechanisms of limb-girdle muscular dystrophy, any definitive treatments do not exist, and current strategies are only based on the improvement of the signs of disorder and to enhance the life quality without resolving an underlying cause. There is a crucial relationship between pharmacological therapy and different consequences; therefore, other treatment strategies will be required. New approaches, such as gene replacement, gene transfer, exon skipping, siRNA knockdown, and anti-myostatin therapy, which can target specific cellular or molecular mechanism of LGMD, could be a promising avenue for the treatment. Recently, genome engineering strategies with a focus on molecular tools such as CRISPR-Cas9 are used to different types of neuromuscular disorders and show the highest potential for clinical translation of these therapies. Thus, recent advancements and challenges in the field will be reviewed in this paper.
    Keywords:  Adeno-associated virus; Exon skipping; Gene editing; Gene therapy; LGMD; Limb-girdle muscular dystrophy
  11. Front Genet. 2019 ;10 1393
    Reyes A, Rusecka J, Tońska K, Zeviani M.
      RNase H1 is able to recognize DNA/RNA heteroduplexes and to degrade their RNA component. As a consequence, it has been implicated in different aspects of mtDNA replication such as primer formation, primer removal, and replication termination, and significant differences have been reported between control and mutant RNASEH1 skin fibroblasts from patients. However, neither mtDNA depletion nor the presence of deletions have been described in skin fibroblasts while still presenting signs of mitochondrial dysfunction (lower mitochondrial membrane potential, reduced oxygen consumption, slow growth in galactose). Here, we show that RNase H1 has an effect on mtDNA transcripts, most likely through the regulation of 7S RNA and other R-loops. The observed effect on both mitochondrial mRNAs and 16S rRNA results in decreased mitochondrial translation and subsequently mitochondrial dysfunction in cells carrying mutations in RNASEH1.
    Keywords:  7S DNA; 7S RNA; RNase H1; mitochondria; mitochondrial disease; mtDNA; transcription; translation
  12. Adv Exp Med Biol. 2020 Feb 19.
    Larijani B, Foroughi Heravani N, Alavi-Moghadam S, Goodarzi P, Rezaei-Tavirani M, Payab M, Gholami M, Razi F, Arjmand B.
      Autism spectrum disorders as a group of pediatric neurodevelopmental diseases is a crucial part of the worldwide disabilities which have influence in communication skills, social interactions, and ability to understand the concepts. The precise pathophysiology of autism spectrum disorders due to the abundance of involved mechanisms is unknown. Some of these involved mechanisms are related to genetic factors, chronic neuro inflammation, mitochondrial dysfunction, oxidative stress, immune dysregulation, hormonal imbalance, and environmental factors. Current main treatments for autisms are behavioral, nutritional and medical therapies, however there is not definitive treatment approach. Therein, more novel therapies are still required to improve the symptoms. Several preclinical and clinical evidence were shown that stem cell therapy is a potential treatment option for autism spectrum disorders individuals. Considering the significant factors which can affect the outcome of stem cell therapeutic effects including stem cell types, route and dosage of administration, and mechanism of activity along with selecting best animal models can be very important in performing clinical trials.
    Keywords:  Autism spectrum disorders; Neuro inflammation; Neurological disorders; Regenerative medicine; Stem cell therapy
  13. Nervenarzt. 2020 Feb 19.
    Lehmann Urban D, Schneider I.
      Gene-specific treatment for hereditary muscle diseases has made great progress in recent years. The pathomechanisms of many of these diseases could be decrypted using molecular genetic techniques, paving the way for disease-modifying treatment options. A milestone was undoubtedly the successful translation of the antisense oligonucleotide (ASO) technology into clinical practice, with gene-specific ASOs being approved for the first time in 2016 for the treatment of spinal muscular atrophy and Duchenne muscular dystrophy. This article reviews recent developments in the field of antisense and gene therapies for hereditary muscle diseases.
    Keywords:  Antisense oligonucleotide; Cell-based treatment; Gene therapy; Muscular dystrophy, Duchenne; Myotonic dystrophy 1
  14. Am J Pathol. 2020 Feb 18. pii: S0002-9440(20)30087-0. [Epub ahead of print]
    McCarron A, Cmielewski P, Reyne N, McIntyre C, Finnie J, Craig F, Rout-Pitt N, Delhove J, Schjenken JE, Chan HY, Boog B, Knight E, Gilmore RC, O'Neal WK, Boucher RC, Parsons D, Donnelley M.
      Animal models of cystic fibrosis (CF) are essential for investigating disease mechanisms and trialling potential therapeutics. This study generated two CF rat models using clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) gene editing. One rat model carries the common human Phe508del (ΔF508) CF transmembrane conductance regulator (CFTR) mutation, whereas the second is a CFTR knockout model. Phenotype was characterized using a range of functional and histological assessments including nasal potential difference to measure electrophysiological function in the upper airways, RNAscope in situ hybridization and quantitative PCR to assess CFTR mRNA expression in the lungs, immunohistochemistry to localize CFTR protein in the airways, and histopathological assessments in a range of tissues. Both rat models revealed a range of CF manifestations including reduced survival, intestinal obstruction, bioelectric defects in the nasal epithelium, histopathological changes in the trachea, large intestine, and pancreas, and abnormalities in the development of the male reproductive tract. The CF rat models presented here will prove useful for longitudinal assessments of pathophysiology and therapeutics.
  15. J Clin Med. 2020 Feb 14. pii: E520. [Epub ahead of print]9(2):
    Law ML, Cohen H, Martin AA, Angulski ABB, Metzger JM.
      : Duchenne muscular dystrophy (DMD) is an X-linked recessive disease resulting in the loss of dystrophin, a key cytoskeletal protein in the dystrophin-glycoprotein complex. Dystrophin connects the extracellular matrix with the cytoskeleton and stabilizes the sarcolemma. Cardiomyopathy is prominent in adolescents and young adults with DMD, manifesting as dilated cardiomyopathy (DCM) in the later stages of disease. Sarcolemmal instability, leading to calcium mishandling and overload in the cardiac myocyte, is a key mechanistic contributor to muscle cell death, fibrosis, and diminished cardiac contractile function in DMD patients. Current therapies for DMD cardiomyopathy can slow disease progression, but they do not directly target aberrant calcium handling and calcium overload. Experimental therapeutic targets that address calcium mishandling and overload include membrane stabilization, inhibition of stretch-activated channels, ryanodine receptor stabilization, and augmentation of calcium cycling via modulation of the Serca2a/phospholamban (PLN) complex or cytosolic calcium buffering. This paper addresses what is known about the mechanistic basis of calcium mishandling in DCM, with a focus on DMD cardiomyopathy. Additionally, we discuss currently utilized therapies for DMD cardiomyopathy, and review experimental therapeutic strategies targeting the calcium handling defects in DCM and DMD cardiomyopathy.
    Keywords:  Serca2a; calcium; dilated cardiomyopathy; gene therapy; heart; mdx; membrane stabilization; muscular dystrophy; oxidative stress; phospholamban
  16. Drug Deliv Transl Res. 2020 Feb 19.
    Haley RM, Gottardi R, Langer R, Mitchell MJ.
      Gene therapy is a powerful tool against genetic disorders and cancer, targeting the source of the disease rather than just treating the symptoms. While much of the initial success of gene delivery relied on viral vectors, non-viral vectors are emerging as promising gene delivery systems for efficacious treatment with decreased toxicity concerns. However, the delivery of genetic material is still challenging, and there is a need for vectors with enhanced targeting, reduced toxicity, and controlled release. In this article, we highlight current work in gene therapy which utilizes the cyclic oligosaccharide molecule cyclodextrin (CD). With a number of unique abilities, such as hosting small molecule drugs, acting as a linker or modular component, reducing immunogenicity, and disrupting membranes, CD is a valuable constituent in many delivery systems. These carriers also demonstrate great promise in combination therapies, due to the ease of assembling macromolecular structures and wide variety of chemical derivatives, which allow for customizable delivery systems and co-delivery of therapeutics. The use of combination and personalized therapies can result in improved patient health-modular systems, such as those which incorporate CD, are more conducive to these therapy types. Graphical abstract.
    Keywords:  Cancer; Combination therapy; Cyclodextrin; Drug delivery; Gene therapy; Hydrogel; Nanoparticle; Nucleic acid delivery; Oncology; Polymer
  17. Colloids Surf B Biointerfaces. 2020 Feb 07. pii: S0927-7765(20)30084-9. [Epub ahead of print]189 110854
    Wang M, Ruan L, Zheng T, Wang D, Zhou M, Lu H, Gao J, Chen J, Hu Y.
      Photothermal therapy emerges as a promising approach in antitumor treatment. A major challenge for conventional photothermal therapy is its unselective hyperthermia distribution within tumor tissues, which leads to detrimental effects on surrounding healthy tissues and compromised therapeutic effectiveness. In this study, a targeted photothermal delivery nanoplatform (P-D-CS-CNTs) was facilely fabricated by decoration of an acidity-labile polyethylene glycol (PEG) derivative onto chitosan nanoparticles encapsulating single-walled carbon nanotubes. P-D-CS-CNTs displayed a good stability in serum at normal physiological pH and convertibility of surface charges upon exposure to tumoral acidic pH, which was attributed to the acidity-triggered dePEGylation. The confocal laser scanning microscopic observations suggested that such surface-convertibility of nanoparticles facilitated tumor cell uptake, endo/lyososomal escape, and enhanced mitochondrial targeting. Furthermore, upon irradiation with an 808 nm laser, P-D-CS-CNTs could sabotage mitochondria with mild hyperthermia, which further induced the ROS burst from damaged mitochondria. The overdosed ROS ultimately resulted in mitochondrial damage and cell death. These findings indicate that the surface-convertible nanoplatform is promising for improved photothermal anticancer therapy.
    Keywords:  Mitochondria targeted delivery; Photothermal therapy; Surface convertible nanoparticles; pH sensitive
  18. Nervenarzt. 2020 Feb 19.
    Feneberg E, Otto M.
      The pathological hallmarks of Alzheimer's disease are aggregation and accumulation of amyloid-β and tau proteins. So far most interventional studies have focused on the removal of the toxic protein products, such as antibody-based immunotherapies targeted against amyloid-β and tau proteins; however, the development of gene therapies targeting gene products involved in the disease has opened up new therapeutic strategies to reduce the development of toxic protein aggregates by inhibiting the translation of pathological Alzheimer genes using antisense oligonucleotides (ASO). This has a timely influence on development of the disease. This article gives an overview of new advances in ASO-based treatment strategies for Alzheimer's disease.
    Keywords:  Amyloid beta; Antibody therapy; Antisense oligonucleotides; Gene therapy; Tau