Molecular Gene Therapy Research Articles

Autosomal Recessive Non-Syndromic Deafness: Is AAV Gene Therapy a Real Chance?

The etiology of sensorineural hearing loss is heavily influenced by genetic mutations, with approximately 80% of cases attributed to genetic causes and only 20% to environmental factors. Over 100 non-syndromic deafness genes have been identified in humans thus far. In non-syndromic sensorineural hearing impairment, around 75-85% of cases follow an autosomal recessive inheritance pattern. In recent years, groundbreaking advancements in molecular gene therapy for inner-ear disorders have shown promising results. Experimental studies have demonstrated improvements in hearing following a single local injection of adeno-associated virus-derived vectors carrying an additional normal gene or using ribozymes to modify the genome. These pioneering approaches have opened new possibilities for potential therapeutic interventions. Following the PRISMA criteria, we summarized the AAV gene therapy experiments showing hearing improvement in the preclinical phases of development in different animal models of DFNB deafness and the AAV gene therapy programs currently in clinical phases targeting autosomal recessive non syndromic hearing loss. A total of 17 preclinical studies and 3 clinical studies were found and listed. Despite the hurdles, there have been significant breakthroughs in the path of HL gene therapy, holding great potential for providing patients with novel and effective treatment.

RAAV-Mediated Cochlear Gene Therapy: Prospects and Challenges for Clinical Application.

Over the last decade, pioneering molecular gene therapy for inner-ear disorders have achieved experimental hearing improvements after a single local or systemic injection of adeno-associated, virus-derived vectors (rAAV for recombinant AAV) encoding an extra copy of a normal gene, or ribozymes used to modify a genome. These results hold promise for treating congenital or later-onset hearing loss resulting from monogenic disorders with gene therapy approaches in patients. In this review, we summarize the current state of rAAV-mediated inner-ear gene therapies including the choice of vectors and delivery routes, and discuss the prospects and obstacles for the future development of efficient clinical rAAV-mediated cochlear gene medicine therapy.

Epithelial-to-Mesenchymal Transition (EMT) and Signal Transducers and Activators of Transcription (STAT3) Are a Novel Therapeutic Strategy for Hepatocellular Carcinoma (HCC) Prognosis

Abstract Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide and is the second most common cancer in Egypt. Poor diagnosis of HCC is correlated with vascular invasion and metastasis. Epithelial-to-mesenchymal transition (EMT) is the main step in the tumor invasion process whereby epithelial cells lose cell polarity with each other and then undergo a dramatic remodeling of the cytoskeleton. Also, EMT plays a pivotal role in metastasis when epithelial cell layers lose cell-cell contacts in tumor progression due to the loss of E-cadherin and increasing the ability of the spread into surrounding tissues. Signal transducers and activators of transcription (STAT) play a different cellular function as signal transducers in the cytoplasm and transcription activators in the nucleus. STAT3 gene plays a crucial role to affect EMT in cancer progression by promoting cell proliferation and survival through its function as a transcription factor of the tumor. The aim of our study is to investigate the gene expression of STAT3 in HCC (HepG-2) cell lines and the expression of cell differentiation and proliferation markers. Western blotting was used to examine the protein expression of STAT3, E-cadherin, and β-catenin signaling. Our results showed that STAT3 expression levels were detected, as well as a significant increase in the expression of proliferation marker as β-catenin and a significant decrease of differentiation marker as E-cadherin protein expression levels in HepG-2 cell lines. Conclusion Our finding provides novel evidence for using a molecular gene therapy as STAT3, which showed an effect on EMT that plays a pivotal role in the prognosis of HCC. The loss of E-cadherin expression is a hallmark of EMT, because β-catenin is associated with the cytoplasmic domain of E-cadherin. We have emphasized the significant role of EMT and STAT3 in HCC progression, which could be a potential application as a novel therapeutic strategy for HCC treatment.

A review of genetic factors contributing to the etiopathogenesis of anorectal malformations.

Anorectal malformation (ARM) is a common congenital anomaly with a wide clinical spectrum. Recently, many genetic and molecular studies have been conducted worldwide highlighting the contribution of genetic factors in its etiology. We summarize the current literature on such genetic factors. Literature search was done using different combinations of terms related to genetics in anorectal malformations. From 2012 to June 2017, articles published in the English literature and studies conducted on human population were included. A paradigm shift was observed from the earlier studies concentrating on genetic aberrations in specific pathways to genome wide arrays exploring single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) in ARM patients. Rare CNVs (including 79 genes) and SNPs have been found to genetically contribute to ARM. Out of disrupted 79 genes one such putative gene is DKK4. Down regulation of CDX-1 gene has also been implicated in isolated ARM patients. In syndromic ARM de novo microdeletion at 17q12 and a few others have been identified. Major genetic aberrations proposed in the pathogenesis of ARM affect members of the Wnt, Hox (homebox) genes, Sonic hedgehog (Shh) and Gli2, Bmp4, Fgf and CDX1 signalling pathways; probable targets of future molecular gene therapy.

Molecular imaging of stem cell therapy in brain tumors: a step towards personalized medicine.

Advances in gene-based medicine since the 1990s have ushered in a new therapeutic strategy of gene therapy for inborn error genetic diseases and also for cancer [1-6]. Personalized treatment strategies using stem, modified or also genetically engineered cells are becoming a reality in clinical medicine. Allogenic or autologous cells can be used for treatment and possibly for early diagnosis of diseases. Hematopoietic, stromal and organ specific stem cells are under evaluation for cell-based therapies, not only for neurological, but also cardiac, pneumological, gynecological, autoimmune and other disorders. Such cell replacement therapy and gene transfer have provided the basis for the development of potentially powerful new therapeutic strategies for a broad spectrum of neurological diseases. However, currently available treatment modalities for brain tumors, including radical surgical resection followed by radiation and chemotherapy, have substantially improved the survival rates [7, 8]; however, a large proportion of patients with brain tumors remain incurable [9]. Therefore, there is substantial need for more effective therapies for patients with malignant brain tumors, and gene therapy targeting brain tumors should fulfill this requirement. Gene therapy for brain tumors includes many therapeutic strategies and these strategies can be subgrouped in two major categories: (i) molecular and (ii) immunologic. The widely used molecular gene therapy approach is suicide gene therapy based on the conversion of non-toxic prodrugs into active anticancer agents via introduction of enzymes, and genetic immunotherapy involves the gene transfer of immune-stimulating cytokines including interleukin (IL)-4, IL-12 and tumor necrosis factor related apoptosis inducing ligand (TRAIL) [10, 11]. For both molecular and immune gene therapy, stem cells can be used as a delivery vehicle of therapeutic genes [12]. Stem cells possess an inherent tumor tropism and have the capacity to target therapeutic genes to tumors, which supports their use as a reliable delivery vehicle to target therapeutic gene products to primary brain tumors and metastatic cancers throughout the brain [13, 14]. The main promise of this emerging technology centers on the potent migratory tropism exhibited by stem cells for disseminated foci of intracranial pathologic findings. This important characteristic, which has been validated in a wide set of preclinical studies, forms a foundation for the use of transplanted stem cell populations as vehicles for the delivery of tumor-toxic molecules to sites of intracranial tumor [9, 14]. The significance of neuronal stem cell (NSC)-based gene therapy for brain tumor is that it is possible to exploit the tumor-tropic Corresponding author: Bernhard Schaller MD, PhD, DSC University of Oradea Romania Phone: 079 666 99 42b E-mail: skull_base-surgery@yahoo.de Editorial

MAJOR CONGENITAL METABOLIC DISORDERS IN THE FIRST 12 YEARS OF LIFE IN 79, 100 CONSECUTIVELY BORN CHILDREN IN QAZVIN PROVINCE

Objective Deficient enzyme activity may cause congenital metabolic defects. These defectsare inherited in an autosomal recessive, autosomal dominant, and X-linkedpatterns. This study was aimed at investigating the occurrence of metabolicdiseases in Qazvin Province. Materials & Methods This cross-sectional study was performed on 79,100 children aged 12 years orless between 2000 and 2010. Clinical manifestations, laboratory findings, and allother essential information were assessed to precisely diagnose the metabolicdiseases. The sorted information on congenital metabolic defects of the patients,information included in a checklist, and data were analyzed usnig SPSS. Results A total of 57 metabolic disorders were recorded. The difference in the prevalenceof metabolic disorders between male (29 cases) and female (28 cases) wasnot statistically significant. The most frequent congenital metabolic disorderamong our patients was phenylketonuria (PKU; 5 per 1,000 cases), and the leastcommon disorder was galactosemia (3 per 1,000 cases). Conclusion Timely detection and management of congenital metabolic disorders canhelp save the affected children. Prenatal screening programs, molecular genetherapy, and counseling for consanguineous marriage can play important rolesin reducing the rate of metabolic disorders in this province. Keywords: Congenital metabolic disorders; prevalence; population; Qazvin

Neural Stem Cell-based Gene Therapy for Brain Tumors

Advances in gene-based medicine since 1990s have ushered in new therapeutic strategy of gene therapy for inborn error genetic diseases and cancer. Malignant brain tumors such as glioblastoma multiforme and medulloblastoma remain virtually untreatable and lethal. Currently available treatment for brain tumors including radical surgical resection followed by radiation and chemotherapy, have substantially improved the survival rate in patients suffering from these brain tumors; however, it remains incurable in large proportion of patients. Therefore, there is substantial need for effective, low-toxicity therapies for patients with malignant brain tumors, and gene therapy targeting brain tumors should fulfill this requirement. Gene therapy for brain tumors includes many therapeutic strategies and these strategies can be grouped in two major categories: molecular and immunologic. The widely used molecular gene therapy approach is suicide gene therapy based on the conversion of non-toxic prodrugs into active anticancer agents via introduction of enzymes and genetic immunotherapy involves the gene transfer of immune-stimulating cytokines including IL-4, IL-12 and TRAIL. For both molecular and immune gene therapy, neural stem cells (NSCs) can be used as delivery vehicle of therapeutic genes. NSCs possess an inherent tumor tropism that supports their use as a reliable delivery vehicle to target therapeutic gene products to primary brain tumors and metastatic cancers throughout the brain. Significance of the NSC-based gene therapy for brain tumor is that it is possible to exploit the tumor-tropic property of NSCs to mediate effective, tumor-selective therapy for primary and metastatic cancers in the brain and outside, for which no tolerated curative treatments are currently available.

Molecular Gene Therapy: Overexpression of the Alternative NADH Dehydrogenase NDI1 Restores Overall Physiology in a Fungal Model of Respiratory Complex I Deficiency

Defects in oxidative phosphorylation lie at the heart of a wide variety of degenerative disorders, cancer, and aging. Here, we show, using the fungal model Podospora anserina, that the overexpression of the native mitochondrial matrix-faced type II NADH dehydrogenase NDI1, paralogue of the human apoptosis inducing factor AIF1, can fully restore all physiological consequences of respiratory complex I deficiency. We disrupted the 19.3-kDa subunit of the complex I catalytic core, orthologue of the human PSST subunit, leading to a complete absence of the complex without affecting the assembly and/or stability of the rest of the respiratory chain. This disruption caused a several-fold life span extension at the expense of both male and female fertility. The effect was generally similar but markedly milder than that caused by defects in the complex III/IV-dependent pathway and not associated with a clear reduction in the steady-state level of mitochondrial reactive oxygen species. Whereas the native expression of NDI1 was sufficient to overcome lethality, only the artificial, constitutive overexpression of NDI1 could fully remedy this deficiency: The latter strikingly restored both life span and fertility to levels indistinguishable from wild type, thus demonstrating its unique potential in molecular gene therapy.

Local delivery system of cytotoxic agents to tumors by focused sonoporation

Recently, ultrasound-targeting microbubble destruction has been employed in molecular gene therapy, and a new potent nonviral gene transfer method known as 'sonoporation' has been developed. We investigated the efficiency of sonoporation toward growth inhibition of human gingival squamous carcinoma cell line, Ca9-22, in vitro and in vivo. The cytotoxicity of bleomycin (BLM) was investigated using flow-cytometric analysis and Hoechst's staining in vitro assay systems. We found that the delivery of BLM by sonoporation induced cytotoxic effect toward Ca9-22 cells in vitro. Our in vivo results showed that tumors nearly disappeared in Ca9-22 cell-implanted nude KSN/slc mice treated with a low dose of BLM followed by sonoporation during the 4-week experimental period. Histological analysis revealed that the cytotoxic effect was mainly apoptosis. We previously reported that the cytolethal distending toxin B (cdtB) from Actinobacillus actinomycetemcomitans, a periodontopathic bacterium, is responsible for cell cycle arrest and apoptosis in vitro. Thus, we used sonoporation to transfect a cdtB-expressing plasmid into Ca9-22 cells and examined cell viability in vitro and in vivo. We found that an administration of cdtB-expressing plasmid followed by sonoporation-induced marked growth inhibition of Ca9-22 cells and apoptotic cells were also observed in vitro and in vivo. These findings suggest that local administration of cytotoxic agents with sonoporation is a useful method for molecular cancer therapy.

Systemic Mannitol-Induced Hyperosmolality Amplifies rAAV2-Mediated Striatal Transduction to a Greater Extent Than Local Co-infusion

Recombinant viral vectors derived from adeno-associated virus serotype 2 (rAAV2) have been investigated as highly effective vehicles for gene transfer to the central nervous system (CNS). Transduction with rAAV2 vectors results in long-term transgene expression in CNS neurons. Optimal injection parameters leading to efficient targeting and spread of the transgene to large neuroanatomical regions are important in molecular gene therapy studies of the CNS. In the present study, we reexamined the effects of both local and systemic administration of mannitol-induced hyperosmolality on facilitation of transgene expression and vector distribution in the CNS. Systemic intraperitoneal administration of mannitol prior to vector administration improved gene transfer to striatal neurons, increasing the total number of transduced cells by 400% and vector distribution by 200%. On the other hand, local coadministration of mannitol in the striatum increased the number of transduced striatal neurons by 25% and had little effect on transduction volume. In conclusion, we have demonstrated that systemic coadministration of mannitol significantly enhances transgene spread of rAAV2 viral delivery in the brain to a much greater degree than local coadministration.

Ribozymes: Applications to Functional Analysis and Gene Discovery

Ribozymes are catalytic RNA molecules that cleave RNAs with high specificity. Since the discovery of these non-protein enzymes, the rapidly developing field of ribozymes has been of particular interest because of the potential utility of ribozymes as tools for reversed genetics. However, despite extensive efforts, the activity of ribozymes in vivo has not usually been high enough to achieve the desirable biological effects. Now, by the use of RNA polymerase III (pol III) promoters, the ribozyme activity in cells has been successfully improved by developing efficient transport systems for the transcripts to the cytoplasm. In addition, it is possible to cleave a specific target RNA in cells by using an allosterically controllable ribozyme or an RNA-protein hybrid ribozyme. These ribozymes are potentially applicable to molecular gene therapy and efficient gene discovery systems. Furthermore, the developed pol III expression system is applicable to the expression of small interfering RNAs (siRNAs). The advantage of such ribozymes over siRNAs is the high specificity of the ribozyme that would not cause interferon responses.

Maxizyme technology.

Ribozymes are small and versatile nucleic acids that can cleave RNAs at specific sites. These molecules have great potential to be used as effective gene-therapeutic agents. However, because of the limitation for cleavable sequences within the target mRNA, in some cases conventional ribozymes have failed to exhibit precise cleavage specificity. A maxizyme is the dimer of minimized ribozymes (minizymes), which can specifically cleave two distinct target sites. The maxizyme also has an allosteric function in that it can form an active conformation and cleave the two target sites only when it recognizes two distinct target sites. We demonstrated previously that an allosterically controllable maxizyme was a powerful tool in the disruption of an abnormal chimeric RNA (bcr-abl) in cells and in mice. Furthermore, more than five custom-designed maxizymes have clearly demonstrated these allosteric functions in vitro and in vivo. Thus, maxizyme technology is not limited to one specific case, but may have broad general applicability in molecular biology and in molecular gene therapy.

Heart Failure: Pathogenesis and Treatment

Chronic heart failure is increasing in prevalence and putting a growing burden on health services. One reason for this is our success in salvaging lives from large myocardial infarction and in improving the outlook for patients with moderate and severe heart failure; another is the ageing of the population. Despite improvements in the prevention of heart failure by new antihypertensive and hypolipidaemic therapies this trend seems set to continue; indeed, a worldwide epidemic of heart failure is beginning as African, Asian and East European countries adopt western habits of diet and lack of physical exercise. Against this background, heart failure care and research are advancing apace; most of the now standard treatments were introduced in the past two decades. At basic level much has been learned from the functioning of isolated myocytes—their interaction with the myocardial cytoskeleton and interstitial tissue—and clinical research has begun to elucidate the complex neurohormonal, immunological and endocrine maladaptations that occur in chronic heart failure. In addition, our knowledge of clinical physiology and pathophysiology at the cellular, organ and whole body level is increasing fast. Lastly, but most importantly, the evidence base in terms of large-scale randomized clinical trials is growing very quickly and has established unequivocal benefit from treatments such as angiotensin converting enzyme inhibitors, beta-blockers and spironolactone. Other treatments that looked promising have been found ineffective. Non-pharmaceutical approaches under development include ventricular assist devices and electrical resynchronization. The years to come will be an era of molecular gene therapy, stem cell therapy and novel small molecules fashioned from knowledge of molecular biology of the failing heart. The weighty tome by Narula and colleagues is thus well timed. Comprehensive but approachable, it is amply illustrated by figures, explanatory tables and cartoons. The format is conventional, moving from basic scientific principles to pathogenic mechanisms, then management. The account of the surgical management of end-stage heart failure is unusually detailed, whereas the overview of medical treatment of heart failure is too brief at 11 pages to do justice to the wealth of data from clinical trials. The main strength of the book is the material on mechanisms. Many of the real experts are here, including Arnold Katz on the cellular and molecular basis of myocardial contraction. The explanation of the current carrying channels of the myocyte is very elegantly presented. The level of detail is sufficient to allow a clinician to grasp basic processes and to envisage where we can next look for progress in our understanding of cardiomyopathy. Conventional ventricular mechanics and haemodynamics are well covered and there is an authoritative chapter by J S Ingwall on the evolving field of myocardial energetics. Insights are presented on the increasingly complex gene pathways for the expression of hypertrophy and the transition from hypertrophy to failure. This leads in well to a more conventional histological classification and description of the cardiomyopathic disorders. Indeed, myopathies dominate, and clinical management of secondary ischaemic cardiac failure is dealt with in less depth. The neurohormonal, immune and endocrine adaptations are well covered but there is little on cytokine activation, so important in the progression of heart failure. Apoptosis gets its own chapter and there are separate chapters on inflammation, matrix remodelling and altered iron channel expression. More clinically oriented chapters cover hypertensive heart disease, the myocardium in diabetes mellitus and infective diseases (including HIV-related cardiomyopathy). Ischaemic heart disease is accorded just one chapter. I recommend this text to those working in heart failure research and to cardiologists who wish to keep abreast of developments in this expanding area. The emphasis is on cardiomyopathic processes at cellular and organic level; in other words, this is not a clinical ‘how-to’ text.

P53 adenoviral vector (Ad-CMV-p53) induced prostatic growth inhibition of primary cultures of human prostate and an experimental rat model.

Benign prostatic hyperplasia (BPH) is the most common proliferative disease affecting men. Numerous minimally invasive technologies are being developed or are currently in use to obviate the need for transurethral surgery. The goal of the present study was to develop a novel molecular based approach for the treatment of BPH using recombinant p53 adenoviral vector. The over-expression of wt-p53 can cause cell apoptosis or cell growth arrest, thus preventing the uncontrolled cell proliferation underlying BPH pathophysiology. Ad-CMV-p53, a replication-deficient recombinant adenovirus containing cytomegalovirus promoter driving p53 gene, was used. Human prostate stromal (PS) cells were evaluated for apoptosis (TUNEL assay), mRNA levels of key cell cycle regulators influencing apoptosis (p-53, Bax and Bcl-2) using quantitative RT-PCR and cytotoxicity after Ad-CMV-p53. Ad-CMV-p53 was unilaterally injected into rat ventral prostates and growth inhibition was measured by prostate weight 3 weeks after injection. In vitro exposure to Ad-CMV-p53 significantly inhibited the proliferation of PS cells, induced mRNA over-expression of both wt-p53 and Bax, and increased the proportion of apoptotic cells. A 30% decrease in average prostate weight was demonstrated in rodents after Ad-CMV-p53 injection. The results suggest that further investigation of molecular gene therapy with recombinant wt-p53 adenovirus for the treatment of BPH is warranted.