Treatment Of Genetic Diseases Research Articles

Study Designs in Genetic Epidemiology

Genetic epidemiology, as a relatively new issue, aims to explore the independent role of genetic-environmental determinants of diseases. Genetic epidemiology studies, depending on the objective, encompass the most preliminary surveys from the attempts to find family history in the occurrence of diseases to the most advanced surveys including specific strategies by clinical trials in the prevention of genetic diseases. Different objectives in genetic epidemiology studies require special methods and study designs. In this review, chief designs including familial aggregation, heritability, segregation, linkage, and association are evaluated; likewise, the purpose of diverse kinds of studies and analyses is briefly discussed. The utilization of study designs and related analyses according to the aims are the main issues and necessary in the accurate implementation of the study. Some methodological issues in relation to studies on tuberculosis are also reported. Attention to these issues might be useful in the implementation of these methods in the studies designed for the prevention and treatment of genetic disorders.

A Reporter-Based Screen to Identify Potent 3’ Trans-Splicing Molecules for Endogenous RNA Repair

In the treatment of genetic disorders, repairing defective pre-mRNAs by RNA trans-splicing has become an emerging alternative to conventional gene therapy. Previous studies have made clear that the design of the binding domains of the corrective RNA trans-splicing molecules (RTMs) is crucial for their optimal functionality. We established a reporter-based screening method that allows for selection of highly functional RTMs from a large pool of variants. The efficiency and functionality of the screen were validated in the COL7A1 gene, in which mutations are the cause of the skin disease dystrophic epidermolysis bullosa. Comparison of RTMs containing different binding domains hybridizing to COL7A1 intron 64/exon 65 revealed highly different trans-splicing efficiencies. Isolated RTMs were then adapted for endogenous trans-splicing in a recessive dystrophic epidermolysis bullosa (RDEB) keratinocyte cell line expressing reduced levels of COL7A1 mRNA. Our results confirm the applicability and relevance of prescreening reporter RTMs, as significant levels of endogenous COL7A1 mRNA repair were seen with RTMs identified as being highly efficient in our screening system.

Medical Genetics: An Overview

Medical genetics is a branch of human genetics confined to studying structure and function of the genetic material in health and disease states of human beings. It comprises studying causes and mechanisms of pathogenesis of genetic disorders, clinical characterization of different types of these disorders and their modes of inheritance, study of diagnostic techniques used in their diagnosis and delineation of effective prophylactic and therapeutic measures resorted to in managing patients and carriers of these diseases. The wide spectrum of medical genetics includes six main fields: basic, clinical, diagnostic, prophylactic, therapeutic and applied genetics. The scopes of most of these fields are self-explanatory with few exceptions. Pathogenetics, a branch of basic genetics, implies the study of mutagens, mutations, pathogenetic mechanisms responsible for development of genetic diseases and anti-mutation mechanisms that protect the genome from the pathological consequences of these mechanisms. Formal genetics, another branch of basic genetics, is concerned with deducing and figuring out relevant genetic data from constructed figures that contain specific genetic information. These informative figures include, for instance, constructed family pedigrees, linkage maps and chromosomal maps. Applied genetics denotes use of knowledge of other fields of medical genetics in many significant applications like genetic counseling, fetal therapy and forensic genetics. Treatment of genetic disorders and prevention of their complications represent the main targets of therapeutic and prophylactic genetics, respectively. The importance of medical genetics resides in hopes of its ability to offer alleviating measures and curative therapies for disprevileged patients with genetic diseases and, more importantly, to succeed in achieving radical preventive approaches of these diseases. This article is intended to offer a simple and concise overview of medical genetics including the various aspects of its different fields.

Chemiluminescence Molecular Detection of Sequence-Specific HBV-DNA Using Magnetic Nanoparticles

Hepatitis B virus is a kind of DNA virus which can cause serious epidemic disease. The analysis and detection of sequence-specific DNA have great significance in forensic analysis, early-stage identification and treatment of genetic disorders. Chemiluminescent detection of DNA has been applied in many fields, such as biological technology and molecular biology, due to its simple operation and high sensitivity. On the other hand, owing to possessing easy magnetic separation and large surface properties, magnetic nanoparticles have also been employed as special carriers to immobilize biomolecules. In this paper, the magnetic nanoparticles are prepared by soft-template method with uniform shape and good dispersion. Then a detection method of hepatitis B virus DNA is established taking advantages of both chemilumiescence with the system of alkaline phosphatase catalyzing 3-(2'-spiroadamantane)- 4-methoxy-4-(3"-phosphoryloxy) phenyl-1, 2-dioxetane and magnetic nanoparticles. The optimization of conditions affecting the hybridization reaction and the chemilumiescence detection are also investigated to promise a high sensitivity.

Non-Viral Delivery of RNA Interference Targeting Cancer Cells in Cancer Gene Therapy

RNA interference (RNAi) is a collection of small RNA-directed mechanisms that result in sequence-specific inhibition of gene expression. RNAi delivery has demonstrated promising efficacy in the treatment of genetic disorders in cancer. Although viral vectors are currently the most efficient systems for gene therapy, potent immunogenicity, mutagenesis, and the biohazards of viral vectors remain their major risks. Various non-viral delivery vectors have been developed to provide a safer approach for gene delivery, including polymers, peptides, liposomes, and nanoparticles. However, some concerns and challenges of these non-viral gene delivery approaches remain to be overcome. In this review, we summarize the recent progress in the development of non-viral systems delivering RNAi and the currently available preclinical and clinical data, and discuss the challenges and future directions in cancer therapy.

Oligomerized alpha-helical KALA peptides with pendant arms bearing cell-adhesion, DNA-binding and endosome-buffering domains as efficient gene transfection vectors

In this study, a number of KALA-based α-helical peptides were designed and synthesized as non-viral gene carriers. The effects of lysine and histidine residues in the pendant arms and cell-binding RGD motif on DNA binding, particle size, zeta potential, cytotoxicity and gene expression efficiency were first explored. Increasing the lysine and histidine residues reduced particle size and increased zeta potential of DNA complexes, leading to greater gene expression efficiency. In addition, the introduction of RGD group further improved gene expression level. The peptide with optimal compositions, RGDN3K6H3CKHLAKALAKALAC (RC29), was then oligomerized to form di-, tri- and tetra-RC29 via disulfide linkage. Upon oligomerization, RC29 attained a 3-dimensional long α-helical structure with pendant arm(s) extending transversally outwards. Each arm contains a cell-adhesion motif (RGD), DNA-binding and endosome-buffering domains. The α-helicity of the oligomerized peptides was evaluated by circular dichroism (CD) spectroscopy, which showed that an increased oligomerization degree led to a stronger α-helical structure. These peptides form complexes with DNA efficiently. The minimum size and maximum zeta potential of tri-RC29/DNA complexes was about 200 nm and 32.5 mV, respectively. In comparison, RC29 formed DNA complexes with a similar zeta potential, but particle size was significantly larger (355 nm). DNA complexes formed at pH 7.0 yielded higher gene expressions than those formed at pH 5.5 and 6.5. Among all the oligomerized peptides, tri-RC29 provided the highest gene expression efficiency, and its peak luciferase level was 1.5 times higher than that yielded by PEI at its optimal N/P ratio (i.e. 10). Moreover, oligomerized RC29/DNA complexes were less cytotoxic than PEI/DNA complexes. These α-helical peptides can be promising carrier for delivery of therapeutic genes in the treatment of genetic disorders.

Gene therapy approaches for lysosomal storage disorders, a good model for the treatment of mendelian diseases

This review describes the different gene therapy technologies applied to approach lysosomal storage disorders, monogenic conditions, with known genetic and biochemical defects, for many of which animal models are available. Both viral and nonviral procedures are described, underlying the specific needs that the treatment of genetic disorders requires. Lysosomal storage disorders represent a good model of study of gene therapeutic procedures that are, or could be, relevant to the treatment of several other mendelian diseases.

Adeno-Associated Virus Activates an Innate Immune Response in Normal Human Cells but Not in Osteosarcoma Cells

Adeno-associated virus (AAV) is a small, DNA-containing dependovirus with promising potential as a gene delivery vehicle. Given the variety of applications of AAV-based vectors in the treatment of genetic disorders, numerous studies have focused on the immunogenicity of recombinant AAV. In general, AAV vectors appear not to induce strong inflammatory responses. We have found that AAV2, when it infects the osteosarcoma cells U2OS, can initiate part of its replicative cycle in the absence of helper virus. This does not occur in untransformed cells. We set out to test whether the cellular innate antiviral defenses control this susceptibility and found that, in nonimmune normal human fibroblasts, AAV2 induces type I interferon production and release and the accumulation of nuclear promyelocytic leukemia bodies. AAV fails to mobilize this defense pathway in the U2OS cells. This permissiveness is in large part due to impairment of the viral sensing machinery in these cells. Our investigations point to Toll-like receptor 9 as a potential intracellular sensor that detects AAV2 and triggers the antiviral state in AAV-infected untransformed cells. Efficient sensing of the AAV genome and the ensuing activation of an innate antiviral response are thus crucial cellular events dictating the parvovirus infectivity in host cells.

Patents on Non-Viral Mediated Gene Delivery

Gene therapy is a promising therapeutic modality for the treatment of genetic disorders. Gene therapy has been able to correct many of the genetic diseases at the root of their cause by systematizing genetic information that encodes for all functions of every cell in our body. Recent studies have identified novel molecular targets for genetic disorders that can be used to deliver gene to the specific site. Gene therapy applications require safe and efficient method for gene transfer. Over the last decade, non-viral and viral gene therapy approaches have been tested in preclinical studies and human clinical trials. Gene delivery via conventional means by using viral vectors has several undesirable side effects such as insertion of mutational viral gene into the host genome and overwhelming immune and inflammatory responses. As compared to viral vectors, non-viral vehicles has received great attention due to their several favorable properties, including low toxicity and immunogenicty, resistance to nuclease, and their high affinity for DNA targets. Here, we describe how non-viral gene-transfer vehicles have been used and can be modified to target specific tissues for gene therapy. This review focuses on existing and emerging patents on non-viral based genetic engineering strategies for the delivery of therapeutic molecules or several approaches for genetic disorder treatment.

Defects in Bile Acid Biosynthesis‐Diagnosis and Treatment

Bile acid synthetic defects represent a specific category of metabolic liver disease. This article highlights the history and summarizes our analytical approach to the diagnosis and treatment of genetic defects in bile acid synthesis. By the application of mass spectrometry as a screening tool, it is possible to perform rapid diagnosis of potential inborn errors in bile acid synthesis from urinary bile acid analysis. Molecular techniques then afford the identification of specific mutations in genes encoding the enzymes responsible for bile acid synthesis. Using this approach, 6 of the 7 known genetic defects that are causes of progressive cholestatic liver disease, syndromes of fat-soluble vitamin malabsorption, or neurological disease, have been characterized. Bile-acid therapy using oral cholic acid has proven effective in most of these bile acid synthetic defects making early diagnosis crucial to optimum clinical prognosis.

Treatment of human disease by adeno-associated viral gene transfer

During the past decade, in vivo administration of viral gene transfer vectors for treatment of numerous human diseases has been brought from bench to bedside in the form of clinical trials, mostly aimed at establishing the safety of the protocol. In preclinical studies in animal models of human disease, adeno-associated viral (AAV) vectors have emerged as a favored gene transfer system for this approach. These vectors are derived from a replication-deficient, non-pathogenic parvovirus with a single-stranded DNA genome. Efficient gene transfer to numerous target cells and tissues has been described. AAV is particularly efficient in transduction of non-dividing cells, and the vector genome persists predominantly in episomal forms. Substantial correction, and in some instances complete cure, of genetic disease has been obtained in animal models of hemophilia, lysosomal storage disorders, retinal diseases, disorders of the central nervous system, and other diseases. Therapeutic expression often lasted for months to years. Treatments of genetic disorders, cancer, and other acquired diseases are summarized in this review. Vector development, results in animals, early clinical experience, as well as potential hurdles and challenges are discussed.

Stimulation of N-Linked Glycosylation and Lipid-Linked Oligosaccharide Synthesis by Stress Responses in Metazoan Cells

ABSTRACTEndoplasmic reticulum (ER) stress responses comprising the unfolded protein response (UPR) are activated by conditions that disrupt folding and assembly of proteins inside the ER lumenal compartment. Conditions known to be proximal triggers of the UPR include saturation of chaperones with misfolded protein, redox imbalance, disruption of Ca2+ levels, interference with N-linked glycosylation, and failure to dispose of terminally misfolded proteins. Potentially, ER stress responses can reprogram cells to correct all of these problems and thereby restore ER function to normal. This article will review literature on stimulation of N-linked glycosylation by ER stress responses, focusing on metazoan systems. The mechanisms involved will be contrasted with those mediating stimulation of N-linked glycosylation by cytoplasmic stress responses. This information will interest readers who study the biological roles of stress responses, the functions of N-linked glycans, and potential strategies for treatment of genetic disorders of N-linked glycosylation.

998. The HIV-1 LTR-hsp70 Fusion Promoter Directs HIV-1 Inducible Expression of Anti-Rev siRNA along with HIV-1 Rev Transdominant Mutant RevM10

Top of pageAbstract Small interfering RNA (siRNA)-mediated knockdown of deleterious transcripts of cellular or viral origin has potential applications for treatment of hereditary diseases, infections and cancer. We describe here a method to further supplement the potency of siRNA by simultaneous delivery of a gene in conjunction with an siRNA from a novel promoter cassette. This technique can be applied to deliver corrective genes and simultaneously knock out deleterious gene expression. The gene in question can be a corrected replacement gene with codon modifications to prevent the siRNA from targeting the corrective gene, in the case of hereditary diseases, or a suicide/transdominant mutant gene employed to further potentiate the anti-tumor or anti-viral effects of siRNA. As proof of principle we have expressed the HIV-1 transdominant mutant revM10 in conjunction with an anti-Rev siRNA from our HIV-1 LTR, Drosophila hsp70 fusion promoter to further potentiate the anti-HIV-1 effect of siRNA and also to offset resistant mutants that may arise from any single approach. HIV-1 inducible siRNA expression or RevM10 expression is observed only when cells are infected with pNL4-3. A potent synergistic response is observed in cells transfected with siRNA-RevM10 construct as compared with cells transfected with siRNA alone. We are in the process of monitoring whether or not the dual cassette reduces the frequency of viral mutants resistant to one or the other antiviral agent when applied alone. The potential applications of this approach in gene therapy settings will be discussed.

Efficient production of human FVIII in hemophilic mice using lentiviral vectors

Lentiviral vectors (LV) have the ability to integrate their proviral DNA containing a therapeutic gene into the host cell's genome. Therefore, these vectors have a great potential for gene therapy especially in the treatment of hereditary diseases like hemophilia A, which require lifelong expression of the transgene. We constructed an HIV-1-based LV containing human B-domain-deleted factor VIII (FVIII) cDNA under the control of a promoter consisting of the chicken β-actin promoter, CMV enhancers, and a large synthetic intron (CAG), which is a robust transcription promoter. High levels of FVIII expression from this vector could be demonstrated in vitro in 293T cells, primary liver cells, and hematopoietic progenitor cells. To test whether this viral vector was able to correct the bleeding disorder of C57BL/6 FVIII knockout mice, we transduced these mice with the FVIII LV either by intraperitoneal injection or by transplantation with transduced syngeneic bone marrow. FVIII production was analyzed in the blood plasma for a period of 3 months; however, only low levels of FVIII (<50 mU), which were below 5% of normal FVIII levels of 1000 mU, could be detected. Further analysis revealed that the low levels of FVIII activity present in the blood plasma were due to the presence of neutralizing antibodies to FVIII and not due to lack of expression of FVIII from the viral vector. FVIII expression could be detected in the tissues of the transduced mice by Western blot analysis and in ex vivo cultures. These data demonstrate that LVs are able to produce therapeutic levels of FVIII in knockout mice when administered by ip infection or by transduced hematopoietic cells. The challenge is to overcome the immune barriers to the therapeutic gene product.

Britain the first to formally ban human cloning

The British government announced in April that it would ban all forms of human reproductive cloning. This makes Britain the first country to do this and comes as part of a package of legislation designed to allay fears regarding misuse of genetic technologies. The health secretary, Alan Milburn, announced the comprehensive ban on human cloning along with a moratorium on the use of genetic data by insurance companies and an increased availability of genetic screening tests on the UK National Health Service. Two new national laboratories for research into diagnostics and treatments for genetic disorders are also to be established. The cloning ban signifies a clear demarcation between the application of stem cell technologies for therapeutic research, which is allowed in Britain, and the use of such technologies in the creation of cloned humans. D.S.

RNA repair: a novel approach to gene therapy

Treatment of genetic disorders by gene therapy has conventionally been attempted through the transfer of a wild type version of a gene to the cells of a patient harboring defective copies of a disease associated gene. Despite significant advances using this paradigm, several technical hurdles must still be overcome before this ‘gene replacement’ approach will become useful in the treatment of a variety of genetic maladies. Such limitations, have led a number of researchers to begin to investigate alternative strategies to genetic therapy. Repair of mutant genetic instructions represents a fundamentally different approach to genetic therapy that may have significant advantages over gene replacement. Herein, we will discuss recent advances using repair of mutant RNAs as a novel means to correct genetic deficiencies.

Fanconi anemia and beta c deficiency-associated pulmonary alveolar proteinosis as two hereditary diseases of childhood which are potentially curable by stem cell gene therapy but require different therapeutic approaches.

Fanconi anemia (FANC) and pulmonary alveolar proteinosis associated with deficiency of the beta-chain common to the GM-CSF/IL3/IL5 receptors (beta c-PAP) are rare inherited disorders of childhood or adolescence. Hematopoietic stem cell gene therapy aiming at reintroducing the wildtype cDNA as a new concept for the treatment of hereditary diseases may be applicable to FANC and PAP, as both disorders can be successfully treated by allogeneic stem cell transplantation. However, there are important distinctions to be made between the two diseases: FANC seems to be a disorder with functional stem cell deficiency. Thus, introduction of the wildtype cDNA should provide an in vivo growth advantage to genetically corrected stem cells so that corrected cells and their progeny may expand in vivo and slowly repopulate the entire hematopoietic system. In beta c-PAP, the defect has no major impact on proliferation or differentiation of stem cells. Therefore, introduction of the wildtype gene will probably not provide any selective growth advantage and the percentage of corrected cells in the hematopoietic compartment depend on the percentage of stem cells initially transduced as the current technology only allows for transduction of stem cells with low efficiency. The introduction of a second selectable cDNA into the vector might be used to provide selective growth for modified cells and thus overcome a low gene transfer efficiency of stem cells. The correction of rare monogenetic diseases may serve as a model for gene therapy prior to attempts to treat more common and complex polygenetic diseases. The studies outlined here will be helpful envisioning new treatment strategies for other inherited monogenetic diseases such as mucopolysaccharidosis, Gauchers disease or adrenoleukodystrophy.

Ribozyme-mediated repair of sickle beta-globin mRNAs in erythrocyte precursors.

Sickle cell anemia is the most common heritable hematological disease, yet no curative treatment exists for this disorder. Moreover, the intricacies of globin gene expression have made the development of treatments for hemoglobinopathies based on gene therapy difficult. An alternative genetic approach to sickle cell therapy is based on RNA repair. A trans-splicing group I ribozyme was used to alter mutant beta-globin transcripts in erythrocyte precursors derived from peripheral blood from individuals with sickle cell disease. Sickle beta-globin transcripts were converted into messenger RNAs encoding the anti-sickling protein gamma-globin. These results suggest that RNA repair may become a useful approach in the treatment of genetic disorders.

Hepatic iron overload: direct HFE (HLA-H) mutation analysis vs quantitative iron assays for the diagnosis of hereditary hemochromatosis.

Among patients with hepatic iron overload, the distinction between hereditary hemochromatosis (HH), a common yet treatable genetic disease, and other causes of siderosis remains problematic. The recent discovery of a specific homozygous mutation (C282Y) in a novel major histocompatibility complex class I-like gene (named HLA-H or HFE) in 80% to 100% of well-characterized cases of HH suggests that direct DNA-based mutation analysis may help resolve this dilemma. To assess the clinical utility of direct HLA-H mutation analysis in a typical diagnostic setting, we measured genotypic and phenotypic parameters of iron overload in 37 subjects with biopsy-proven hepatic siderosis (2+ or greater) and in 127 healthy control subjects. The prevalence of C282Y homozygotes was significantly greater in the hepatic siderosis group (32%) than in the control group (0%), confirming the association between this homozygous mutation and hepatic iron overload. In the hepatic siderosis group, C282Y homozygotes had significantly higher hepatic iron and ferritin levels, a significantly lower prevalence of hepatitis C virus or alcoholic liver disease, but no significant difference in the saturation of serum transferrin. Of the 20 subjects with a hepatic iron index (HII) in the previously defined "hemochromatosis range" (>1.9), 9 (45%) were C282Y homozygotes. Of the 11 nonhomozygous subjects with an HII greater than 1.9 (presumed false-positive HIIs), 10 (91%) had hepatic cirrhosis compared with 3 of 9 (33%) homozygotes with an HII greater than 1.9 who had cirrhosis (P<.02). The HII thus has poor diagnostic specificity for predicting genotypic HH in patients with cirrhosis. We conclude that direct determination of the HLA-H C282Y genotype may be the single best diagnostic test for HH, particularly in patients with cirrhosis, for whom the HII is quite nonspecific.

The role of nitric oxide in limiting gene transfer: parallels to viral host defenses.

Despite immense efforts, gene therapy remains an elusive goal for the chronic treatment of genetic disorders like cystic fibrosis. The most popular approach is to use adenovirus-derived vectors because they can infect replicating as well as terminally differentiated cells and there is little risk of insertional mutations because the adenovirus DNA does not incorporate into the host genome. However, treatment of cystic fibrosis with adenovirus vectors has met with little success as cellular and humoral immune responses limit the repeated use of adenovirus vectors. Little is known about the mechanisms of host inflammatory defenses against viral infections and adenovirus gene therapy is likely to be limited by these host defenses. In this issue of AJRCMB, the study by Haddad and associates (1) shows that nitric oxide greatly reduces the efficiency of adenovirus expression in lung. Shortly after nitric oxide was identified as the endogenous endothelialderived relaxing factor, activated macrophages were shown to induce a distinct isozyme of nitric oxide synthase and produce large amounts of nitric oxide. It soon became apparent that tumor necrosis factor, interferons, and a variety of cytokines cause almost every cell type and tissue in the body to induce the macrophage isoform of nitric oxide synthase to produce micromolar concentrations of nitric oxide. In pioneering work on the metabolic production of nitrate in humans, Green and Tannenbaum (2) showed that healthy male volunteers suffering from colds show an enormous increase in nitrate in urine. The overproduction of nitric oxide is a major contributor to the fall of blood pressure in septic shock. For the host to dramatically induce a potentially toxic species like nitric oxide as part of the inflammatory response, there must have been an overwhelming evolutionary pressure favoring some of the actions of nitric oxide. One of the most reasonable hypotheses put forward to date is that nitric oxide strongly depresses viral replication in tissues (3). One action of nitric oxide is to reversibly inhibit ribonucleotide reductase, a key enzyme necessary for the synthesis of deoxyribonucleotides in DNA (4). This would not affect the adenovirus vectors since the gene vector is already defective in replication. However, nitric oxide