DOCSLIB.ORG

Widespread Gene Transfection Into the Central Nervous System of Primates

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Widespread Gene Transfection Into the Central Nervous System of Primates Gene Therapy (2000) 7, 759–763 2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt NONVIRAL TRANSFER TECHNOLOGY RESEARCH ARTICLE Widespread gene transfection into the central nervous system of primates Y Hagihara1, Y Saitoh1, Y Kaneda2, E Kohmura1 and T Yoshimine1 1Department of Neurosurgery and 2Division of Gene Therapy Science, Department of Molecular Therapeutics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan We attempted in vivo gene transfection into the central ner- The lacZ gene was highly expressed in the medial temporal vous system (CNS) of non-human primates using the hem- lobe, brainstem, Purkinje cells of cerebellar vermis and agglutinating virus of Japan (HVJ)-AVE liposome, a newly upper cervical cord (29.0 to 59.4% of neurons). Intrastriatal constructed anionic type liposome with a lipid composition injection of an HVJ-AVE liposome–lacZ complex made a similar to that of HIV envelopes and coated by the fusogenic focal transfection around the injection sites up to 15 mm. We envelope proteins of inactivated HVJ. HVJ-AVE liposomes conclude that the infusion of HVJ-AVE liposomes into the containing the lacZ gene were applied intrathecally through cerebrospinal fluid (CSF) space is applicable for widespread the cisterna magna of Japanese macaques. Widespread gene delivery into the CNS of large animals. Gene Therapy transgene expression was observed mainly in the neurons. (2000) 7, 759–763. Keywords: central nervous system; primates; gene therapy; HVJ liposome Introduction ever, its efficiency has not been satisfactory in vivo. Recently HVJ-AVE liposome, a new anionic-type lipo- Despite the promising results in experimental animals, some with the envelope that mimics the human immuno- gene therapy has, so far, not been successful in clinical deficiency virus (HIV), has been developed.13 Based upon 1 situations. As Leiden pointed out, many gene delivery an in vivo trial, this vector has a higher efficiency of gene systems show only limited transgene expression in expression than conventional HVJ liposomes and is humans, unlike those in rodents. Difference in the trans- expected as one of the most useful tools for in vivo gene gene expression between humans and rodents might be therapy. We previously tried the intrathecal adminis- responsible for the disparity and thus, more representa- tration of HVJ-AVE liposome-bcl-2 complex in rats, tive conditions of clinical situations are needed to evalu- which successfully resulted in a diffuse gene transfection ate the strategies for gene therapy. The non-human pri- into the entire CNS.14 mate is the most suitable subject for preclinical studies of In the present study, the efficiency of HVJ-AVE lipo- human gene therapy. Therefore, we evaluated the extent somes in the transfer of DNA into monkey CNS was of gene transfection into the central nervous system evaluated by intrathecal injection through the cisterna (CNS) in non-human primate. magna in three Japanese macaques (Nos. 1, 2 and 3). Gene transfection into the CNS using viral and chemi- Widespread gene transfection was seen over the CNS. A cal gene delivery vectors has been investigated for the fourth Japanese macaque (No. 4) received the ‘empty’ purpose of protection against neuronal insults and HVJ-AVE liposome in the same manner to serve as a con- 2 degeneration. Infection with adenovirus (Ad), adeno- trol. These results were compared with those obtained 3 4–6 associated virus (AAV), herpes simplex virus1 or len- by stereotactic injection into the striatum of two Japanese 7,8 tivirus have resulted in the reasonable extent of gene macaques (Nos 5 and 6), which showed focal gene transfection in the target regions of the CNS. expression around the injection site. Both methods Chemical gene delivery reagents including conven- seemed to be useful for CNS gene therapy. tional hemagglutinating virus of Japan (HVJ) liposomes and a cationic liposome–DNA complex have also been developed.9,10 This liposome-mediated gene delivery sys- Results tem has advantages over viral vectors because they can transfer expression cassettes of unlimited size. They do Injection of HVJ-AVE liposome-lacZ into the cisterna not replicate or recombine to form an infectious agent, magna (Table 1, Figure 1) and may not evoke the inflammatory or immune In monkeys 1, 2 and 3, ␤-gal-positive cells were seen in 11,12 responses as reported in adenoviral vectors. How- many parts of the brain. The areas that exhibited the high transfection rates (mean ± s.d.) were: dentate nucleus (59.4 ± 9.1%), Purkinje cells of cerebellar vermis (47.9 ± Correspondence: Y Hagihara 17.8%), trigeminal nucleus (45.6 ± 18.7%), cochlear Received 13 September 1999; accepted 14 January 2000 nucleus (44.1 ± 0.8%), oculomotor nucleus (43.0 ± 7.9%), Primate CNS gene transfection Y Hagihara et al 760 Table 1 Transfection rate while limited gene expression was detected in more dis- tant areas, where the liposome vehicles rarely reached. LacZ (%) In monkey 4, ␤-gal positive cells were absent. In the injection of HVJ-AVE liposomes into the cisterna Frontal cortex 0.67 ± 1.55 magna of monkey CNS, we did not detect any lympho- Caudate nucleus 9.47 ± 5.42 cyte infiltration and neural cell death. Hippocampus 37.17 ± 1.89 Putamen 2.97 ± 1.16 Parietal cortex 0 Stereotactic injection (Figures 2 and 3) Occipital cortex 0 In monkeys 5 and 6, which received HVJ-AVE liposomes Temporal cortex 0 stereotactically into the striatum, the high lacZ expressing Cerebeller cortex 4.2 ± 2.9 ± ± cells were observed at the injected site (58.5 7.1%; mean Cerebeller vermis 47.87 17.82 ± s.d.). The ␤-gal positive cells were noted in areas more Oculomotor N 43.0 ± 7.93 Dentate nucleus 59.4 ± 9.06 than 10 mm rostral or caudal from the injection tract Cochlear nucleus 44.13 ± 0.81 (Figure 2). High transgene expression was also observed Trigeminal N 45.57 ± 18.73 in the frontal cortex. The ␤-gal-positive cells were rarely Facial nucleus 29.0 ± 2.69 recognized in the temporal, parietal and occipital cortex, ± Olive nucleus 42.13 21.67 which were farther than 15 mm from the tract. Choroid plexus 100 Despite the high transgene expression in the frontal ␤ cortex around the needle insertion, gene expression in the Labeling index of -gal in the CNS after an intrathecal injection of striatum was lower than expected (9.5 ± 5.4%; mean ± HVJ-AVE liposome-lacZ (mean ± s.d.%, n = 5). s.d.). The small leakage of the liposome mixture seen at the injection site might have been responsible for this lim- olive nucleus (42.1 ± 21.7%), hippocampus (37.2 ± 1.9%) ited extent of expression. and facial nucleus (29.0 ± 2.7%). The following areas exhi- bited only limited or no transgene expression: caudate Immunohistochemistry (Figure 4) nucleus (9.5 ± 5.4%), cerebellar cortex (4.2 ± 2.9%), Approximately 90 ± 6% (mean ± s.d.) of the ␤-gal-positive putamen (3.0 ± 1.2%), frontal cortex (0.6 ± 1.6%), parietal cells were stained with anti-neurofilament antibody, cortex and occipital cortex (0%). High transgene which are considered as terminally differentiated neu- expression was observed in the regions close to the cis- rons. We could not identify any GFAP-positive cells terna magna, where the HVJ-AVE liposome was applied, among cells exhibiting blue staining in the presence of a d g b e h c f i Figure 1 ␤-Gal expression in the brain after an intrathecal injection of HVJ-AVE liposome-lacZ complex. Broad ␤-gal expression was seen with X-gal staining (a, b, d, e, g and h). No ␤-gal expression was seen in the frontal cortex (i). The intrathecal injection of HVJ-AVE liposome containing no DNA resulted in no ␤-gal-positive cells (c, f). (a) Cerebellar vermis. Blue stained Purkinje cells were seen among the granular cell layer (×10). (b) Dentate nucleus (×20). (c) Cerebellar vermis, control study (administration with HVJ-AVE liposome carrying no DNA). (d) Hippocampus (×4). (e) Hippocampus (×40). (f) Hippocampus, control study (administration of HVJ-AVE liposome carrying no DNA (×4). (g) Upper cervical cord (×4). (h) Upper cervical cord (×10). (i) Frontal cortex (×4). Gene Therapy Primate CNS gene transfection Y Hagihara et al a 761 a d b e b c f Figure 2 Stereotactic injection of HVJ-AVE liposome–lacZ complex into the striatum assayed with X-gal (a–f). (a) Dense ␤-gal expression at the cortical incision (×4). (b) Refluxed HVJ-AVE liposome–lacZ was diffused and transfected into the cortex from the brain surface (×20). (c) ␤-Gal- positive cells were seen in the deep cortical neurons, which suggests that the HVJ-AVE liposome penetrated into the cortex (×4). (d) High ␤-gal expression at the injection tract (×4). T: injection tract. (e) High ␤-gal expression at the injection site (×20). (f) Diffusion of ␤-gal expressing cells in the white mater (×4). *: injection site. Figure 4 Immunohistochemistry. Staining with anti-neurofilament anti- body (a ×40) and anti-GFAP antibody (c ×10). Most of the ␤-gal express- ing cells were positively stained with anti-neurofilament antibody. None of the ␤-gal expressing cells were positive for anti-GFAP antibody staining. The same procedure with HVJ-cationic liposome was examined by Mabuchi et al15 in an experimental mouse with glioma. Only disseminated glioma cells in the cere- brospinal fluid space expressed the lacZ gene. LacZ trans- fection was absent in either the neuronal or glial cells. This difference between HVJ-AVE and HVJ-cationic lipo- somes may result from the inability of cationic liposomes to penetrate tissues because of both net positive charge and large size (approximately 700 nm in diameter).13 In Figure 3 Transfection efficiency by the distance from the injection site.
Load more

Recommended publications
  • Rnai in Primary Cells and Difficult-To-Transfect Cell Lines
    Automated High Throughput Nucleofection® RNAi in Primary Cells and Difficult-to-Transfect Cell Lines Claudia Merz, Bayer Schering Pharma AG, Berlin, Germany; Andreas Schroers, amaxa AG, Cologne, Germany; Eric Willimann, Tecan AG, Männedorf, Switzerland. Introduction Materials & Methods - Workflow Using primary cells for RNAi based applications such as target identification or – validation, requires a highly efficient transfection displaying the essential steps of the automated Nucleofector® Process: technology in combination with a reliable and robust automation system. To accomplish these requirements we integrated the amaxa 1. Transfer of the cells to the Nucleocuvette™ plate, 96-well Shuttle® in a Tecan Freedom EVO® cell transfection workstation which is based on Tecan’s Freedom EVO® liquid handling 2. Addition of the siRNA, (Steps 1 and 2 could be exchanged), platform and include all the necessary components and features for unattended cell transfection. 3. Nucleofection® process, 4. Addition of medium, Count Cells 5. Transfer of transfected cells to cell culture plate for incubation ® Nucleofector Technology prior to analysis. Remove Medium The 96-well Shuttle® combines high-throughput compatibility with the Nucleofector® Technology, which is a non-viral transfection method ideally suited for primary cells and hard-to-transfect cell lines based on a combination of buffers and electrical parameters. Nucleocuvette Plate Add Nucleofector +– The basic principle and benefits of the (empty) Solution Cell of interest Gene of interest Nucleofector®
    [Show full text]
  • Recombinant DNA and Elements Utilizing Recombinant DNA Such As Plasmids and Viral Vectors, and the Application of Recombinant DNA Techniques in Molecular Biology
    Fact Sheet Describing Recombinant DNA and Elements Utilizing Recombinant DNA Such as Plasmids and Viral Vectors, and the Application of Recombinant DNA Techniques in Molecular Biology Compiled and/or written by Amy B. Vento and David R. Gillum Office of Environmental Health and Safety University of New Hampshire June 3, 2002 Introduction Recombinant DNA (rDNA) has various definitions, ranging from very simple to strangely complex. The following are three examples of how recombinant DNA is defined: 1. A DNA molecule containing DNA originating from two or more sources. 2. DNA that has been artificially created. It is DNA from two or more sources that is incorporated into a single recombinant molecule. 3. According to the NIH guidelines, recombinant DNA are molecules constructed outside of living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell, or molecules that result from their replication. Description of rDNA Recombinant DNA, also known as in vitro recombination, is a technique involved in creating and purifying desired genes. Molecular cloning (i.e. gene cloning) involves creating recombinant DNA and introducing it into a host cell to be replicated. One of the basic strategies of molecular cloning is to move desired genes from a large, complex genome to a small, simple one. The process of in vitro recombination makes it possible to cut different strands of DNA, in vitro (outside the cell), with a restriction enzyme and join the DNA molecules together via complementary base pairing. Techniques Some of the molecular biology techniques utilized during recombinant DNA include: 1.
    [Show full text]
  • REVIEW Gene Therapy
    Leukemia (2001) 15, 523–544 2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu REVIEW Gene therapy: principles and applications to hematopoietic cells VFI Van Tendeloo1,2, C Van Broeckhoven2 and ZN Berneman1 1Laboratory of Experimental Hematology, University of Antwerp (UIA), Antwerp University Hospital (UZA), Antwerp; and 2Laboratory of Molecular Genetics, University of Antwerp (UIA), Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), Antwerp, Belgium Ever since the development of technology allowing the transfer Recombinant viral vectors of new genes into eukaryotic cells, the hematopoietic system has been an obvious and desirable target for gene therapy. The last 10 years have witnessed an explosion of interest in this Biological gene transfer methods make use of modified DNA approach to treat human disease, both inherited and acquired, or RNA viruses to infect the cell, thereby introducing and with the initiation of multiple clinical protocols. All gene ther- expressing its genome which contains the gene of interest (= apy strategies have two essential technical requirements. ‘transduction’).1 The most commonly used viral vectors are These are: (1) the efficient introduction of the relevant genetic discussed below. In each case, recombinant viruses have had material into the target cell and (2) the expression of the trans- gene at therapeutic levels. Conceptual and technical hurdles the genes encoding essential replicative and/or packaging pro- involved with these requirements are still the objects of active teins replaced by the gene of interest. Advantages and disad- research. To date, the most widely used and best understood vantages of each recombinant viral vector are summarized in vectors for gene transfer in hematopoietic cells are derived Table 1.
    [Show full text]
  • Gene Therapy and Genetic Engineering: Frankenstein Is Still a Myth, but It Should Be Reread Periodically
    Indiana Law Journal Volume 48 Issue 4 Article 2 Summer 1973 Gene Therapy and Genetic Engineering: Frankenstein is Still a Myth, but it Should be Reread Periodically George A. Hudock Indiana University - Bloomington Follow this and additional works at: https://www.repository.law.indiana.edu/ilj Part of the Genetics and Genomics Commons Recommended Citation Hudock, George A. (1973) "Gene Therapy and Genetic Engineering: Frankenstein is Still a Myth, but it Should be Reread Periodically," Indiana Law Journal: Vol. 48 : Iss. 4 , Article 2. Available at: https://www.repository.law.indiana.edu/ilj/vol48/iss4/2 This Article is brought to you for free and open access by the Law School Journals at Digital Repository @ Maurer Law. It has been accepted for inclusion in Indiana Law Journal by an authorized editor of Digital Repository @ Maurer Law. For more information, please contact [email protected]. GENE THERAPY AND GENETIC ENGINEERING: FRANKENSTEIN IS STILL A MYTH, BUT IT SHOULD BE REREAD PERIODICALLY GEORGE A. HUDOCKt Biotechnology and the law are far removed from each other as disciplines of human intellect. Yet the law and my own discipline, genetics, have come together in many courtrooms concerning such matters as paternity, and they will continue to intersect with increasing frequency as the visions of 100 years ago become the reality of today. This article examines the implications of recent research for human genetic therapy and genetic engineering, and suggests some guidelines for legal regulation of genetic technology. The following discussion derives from three premises which I view as basic: (1) that which is currently possible in genetic engineering, and in fact has already been done, is generally underestimated; (2) what may be possible in the near future is quite commonly overesti- mated; (3) regulation of the application of genetic technology is possible and will not be overwhelmingly complicated.
    [Show full text]
  • Intramuscular Electroporation Delivery of IFN- Gene Therapy for Inhibition of Tumor Growth Located at a Distant Site
    Gene Therapy (2001) 8, 400–407 2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt RESEARCH ARTICLE Intramuscular electroporation delivery of IFN-␣ gene therapy for inhibition of tumor growth located at a distant site S Li, X Zhang, X Xia, L Zhou, R Breau, J Suen and E Hanna Department of Otolaryngology/Head and Neck Surgery, University of Arkansas School of Medicine, 4001 W Capital Avenue, Little Rock, AR 72205, USA Although electroporation has been shown in recent years to 2 or endostatin gene, also delivered by electro-injection. The be a powerful method for delivering genes to muscle, no increased therapeutic efficacy was associated with a high gene therapy via electro-injection has been studied for the level and extended duration of IFN-␣ expression in muscle treatment of tumors. In an immunocompetent tumor-bearing and serum. We also discovered that the high level of IFN-␣ murine model, we have found that delivery of a low dose of expression correlated with increased expression levels of reporter gene DNA (10 ␮g) to muscle via electroporation the antiangiogenic genes IP-10 and Mig in local tumor under specific pulse conditions (two 25-ms pulses of 375 tissue, which may have led to the reduction of blood vessels V/cm) increased the level of gene expression by two logs of observed at the local tumor site. Delivery of increasing doses magnitude. Moreover, administration of 10 ␮g of interferon (10–100 ␮g) of IFN-␣ plasmid DNA by injection alone did (IFN)-␣ DNA plasmid using these parameters once a week not increase antitumor activity, whereas electroporation for 3 weeks increased the survival time and reduced squam- delivery of increasing doses (10–40 ␮g) of IFN-␣ plasmid ous cell carcinoma (SCC) growth at a distant site in the DNA did increase the survival time.
    [Show full text]
  • HD-Guidance Document Gene Therapy/GMO Environmental Data
    HD-Guidance document Gene Therapy/GMO Environmental Data Guidance document for the compilation of the documentation on possible risks for humans and the environment in support of applications for the authorisation to carry out clinical trials of somatic gene therapy and with medicines containing genetically modified microorganisms (environmental data) In accordance with Articles 22, 35 and Annex 4 ClinO by Swiss Agency for Therapeutic Products (Swissmedic), Federal Office for Public Health (FOPH), Federal Office for the Environment (FOEN), Swiss Expert Committee for Biosafety (SECB) Contents 1 Summary .....................................................................................................................................2 2 Decision tree regarding the compilation of environmental data documentation ................... 3 3 Preliminary remarks / legal basis ..............................................................................................4 4 Definition of genetically modified microorganisms (GMOs) ....................................................5 5 Environmental data documentation ..........................................................................................5 5.1 General information ............................................................................................................................... 5 5.2 Determining and evaluating the risks for humans and the environment (risk assessment) ......... 6 5.2.1 Guidance for the risk assessment ...................................................................................................
    [Show full text]
  • Food and Drug Law Journal
    FOOD AND DRUG LAW JOURNAL EDITOR IN CHIEF Judy Rein EDITORIAL ADVISORY BOARD CHAIR VICE CHAIR FACULTY ADVISOR Laurie Lenkel Robert Giddings Joseph A. Page FDA – OC Hutchison PLLC Georgetown University Law Center ________________________________ Anthony Anscombe James Flaherty Francis Palumbo Sedgwick LLP Fresenius Medical University of Maryland School of Pharmacy Peter Barton Hutt Abraham Gitterman Covington & Burling Arnold & Porter LLP Sandra Retzky FDA – CTP Barbara Binzak Kimberly Gold Blumenfeld Norton Rose Fulbright Joan Rothenberg Buchanan Ingersoll & LLP FDA - CFSAN Rooney PC John Johnson Jodi Schipper Catherine Clements FDA Imports FDA – CDER Express Scripts Alan Katz Christopher van Gundy Kellie Combs toXcel, LLC Keller and Heckman Ropes & Gray LLP Sara Koblitz James Woodlee Nathan Cortez Fish & Richardson Kleinfeld Kaplan & Becker LLP Southern Methodist University Valerie Madamba Emily Wright Blue Apron Pfizer Brian Dahl Dahl Compliance Alan Minsk Kimberly Yocum Consulting LLC Arnall Golden Gregory TC Heartland LLC LLP Sandra dePaulis Lowell Zeta FDA – CVM Nicole Negowetti Hogan Lovells The Good Food Ian Fearon Institute Patricia Zettler British American Tobacco Georgia State James O’Reilly University Law School University of Cincinnati OFFICERS OF THE FOOD AND DRUG LAW INSTITUTE CHAIR: Allison M. Zieve, Public Citizen Litigation Group VICE CHAIR: Jeffrey N. Gibbs, Hyman, Phelps & McNamara, P.C. TREASURER: Frederick R. Ball, Duane Morris LLP GENERAL COUNSEL/SECRETARY: Joy J. Liu, Vertex Pharmaceuticals IMMEDIATE PAST CHAIR: Sheila Hemeon-Heyer, Heyer Regulatory Solutions LLC PRESIDENT & CEO: Amy Comstock Rick GEORGETOWN UNIVERSITY LAW CENTER STUDENT EDITOR IN CHIEF Dana Shaker STUDENT MANAGING EDITORS Jacob Klapholz Christine Rea STUDENT NOTES EDITOR SYMPOSIUM EDITOR Lauren Beegle Alexander P.
    [Show full text]
  • The Art of Transfection (Poster / Pdf)
    TRANSDUCTION NON-VIRAL TRANSFECTION Transduction is the process of using vectors including retroviruses, lentiviruses, adenoviruses, PACKAGE DELIVERY: Chemical Chemical transfection adeno-associated viruses, or hybrids to deliver genetic payloads into cells. Generally, a plasmid transfection reagent containing mRNA carrying genes flanked by viral sequences is first transfected into a producer cell with other reagent virus-associated (packaging) plasmids. In the producer cells, virions form that contain the gene The Art of Transfection of interest. For safety, no plasmid used in the process contains all of the necessary sequences Inserting genetic material into mammalian and insect cells without killing them can be a challenge, CHEMICAL TRANSFECTION for virion formation, and only the plasmid carrying the gene of interest contains signals that but scientists have developed several ways to perform this intricate task. Transfection is the process of Functional proteins or allow it to be packaged into virions. Researchers then extract, purify, and use the virions from Complexation structural components released Chemical carriers represent the most straightforward and widespread tools for gene delivery the producer cells to insert DNA into other cells to stably or transiently express the DNA of introducing nucleic acids (plasmid DNA or messenger, short interfering, or micro RNA) into a cell. from cell or into cytoplasm experiments in mammalian cells. Chemical transfection experiments follow a simple workflow and interest. The transferred genetic material, which lacks viral genes, cannot generate new viruses. Researchers accomplish this with nonviral methods (chemical or physical transfection), or with viral provide high efficiency nucleic acid delivery for the most commonly used cells as well as many methods, commonly referred to as transduction.
    [Show full text]
  • Askbio Announces IND for LION-101, a Novel Investigational AAV Gene
    AskBio Announces IND for LION-101, a Novel Investigational AAV Gene Therapy for the Treatment of Limb-Girdle Muscular Dystrophy Type 2I/R9 (LGMD2I/R9), Cleared to Proceed by U.S. FDA -- LGMD2I/R9 is a Rare Form of Muscular Dystrophy with No Approved Therapies – -- First-in-Human Phase 1/2 Clinical Study Expected to Begin Dosing in 1H 2022 – Research Triangle Park, N.C. – May 25, 2021 – Asklepios BioPharmaceutical, Inc. (AskBio), a wholly owned and independently operated subsidiary of Bayer AG, announced that the U.S. Food & Drug Administration (FDA) has cleared its Investigational New Drug (IND) application for LION-101 to proceed in a Phase 1/2 clinical study. LION-101 is a novel recombinant adeno-associated virus (rAAV) based vector being developed as a one-time intravenous infusion for the treatment of patients with Limb-Girdle Muscular Dystrophy Type 2I/R9 (LGMD2I/R9). LION-101 will be evaluated in a first-in-human Phase 1/2 multicenter study to evaluate a single intravenous (IV) infusion in adult and adolescent subjects with genotypically confirmed LGMD2I/R9. AskBio plans to initiate dosing for the LION-101 Phase 1/2 clinical study in the first half of 2022. “In preclinical mouse models, LION-101 therapy demonstrated both dose-dependent efficacy and tolerability, providing a clear approach to study this novel AAV vector in first-in-human trials,” said Katherine High, MD, President, Therapeutics, AskBio. “Currently there are no approved therapies for LGMD2I/R9, and with limited treatment options that only address symptoms of the disease, the patient burden is profound.
    [Show full text]
  • Human Gene Therapy (Part 6 Of
    Background on Genetic Diseases 13 Background on genetic diseases Chromosomes and inheritance ual. The relative importance of genetic and envi- ronmental influences varies in both patients and Higher organisms package their DNA into seg- diseases. Some medical conditions, such as auto- ments called chromosomes. Each chromosome is mobile accidents or war wounds, may have large composed of one very long stretch of DNA that environmental and very small genetic contribu- is bound to various proteins and other molecules. tions. Most diseases have a mixture of genetic and There are two copies of each of 22 chromosomes environmental contributions (Harsanyi, 1981). In in the cells of a human. In addition, there are two several disorders, such as Huntington or Tay- sex chromosomes. Females have two ‘(X” chromo- Sachs diseases, the influence of a single gene is somes and males have one “X” and one “Y. ” In nor- so large that the disorders are called genetic mal human cells, therefore, there are 46 chromo- diseases. somes: 2 sex chromosomes and 2 copies of each of 22 other chromosomes (these non-sex chromo- somes are called autosomes). SINGLE GENE TRAITS, OR MENDELIAN TRAITS When traits or diseases are primarily deter- The 46 discrete aggregates of DNA and attached mined by a single gene, they obey the relatively protein that comprise the chromosomes are main- simple laws of inheritance first specified by tained inside the nucleus of somatic cells. In germ Gregor Mendel, a monk who lived in the last cen- cells, in contrast, a specialized phenomenon called tury and whose interests in agriculture led him meiosis takes place.
    [Show full text]
  • Micro-RNA Modulation of Insect Virus Replication Verna Monsanto-Hearne and Karyn N
    Micro-RNA Modulation of Insect Virus Replication Verna Monsanto-Hearne and Karyn N. Johnson* School of Biological Sciences, University of Queensland, Brisbane, Australia. *Correspondence: [email protected] htps://doi.org/10.21775/cimb.034.061 Abstract Saleh, 2012; Xu and Cherry, 2014; Mussabekova Te outcome of virus infection in insects is impacted et al., 2017). Many molecular components medi- by regulation of both host and virus gene expres- ate and are mediated by this host–virus cross-talk, sion. A class of small RNAs called micro-RNAs including microRNAs. (miRNA) have emerged as important regulators of microRNAs (miRNAs) are a large class of highly gene expression that can infuence the outcome of conserved, ≈ 22 nt non-coding RNAs that regulate virus infection. miRNA regulation occurs at a com- gene expression. Compared to the more upstream paratively late stage of gene expression, allowing for regulatory mechanisms such as transcriptional rapid control and fne-tuning of gene expression regulation and chromatin remodelling, regulation levels. Here we discuss the biogenesis of miRNAs by miRNA occurs at a later stage of gene expres- from both host and virus genomes, the interactions sion, allowing for rapid control and fne-tuning of that lead to regulation of gene expression, and the gene expression levels (Chen et al., 2013). Comple- miRNA–mRNA interactions that lead to either mentary binding of at least the seed region (second antivirus or provirus consequences in the course of to eighth nucleotide from the 5′-end) of the ≈ 22 nt virus
    [Show full text]
  • The Landscape of Non-Viral Gene Augmentation Strategies for Inherited Retinal Diseases
    International Journal of Molecular Sciences Review The Landscape of Non-Viral Gene Augmentation Strategies for Inherited Retinal Diseases Lyes Toualbi 1,2, Maria Toms 1,2 and Mariya Moosajee 1,2,3,4,* 1 UCL Institute of Ophthalmology, London EC1V 9EL, UK; [email protected] (L.T.); [email protected] (M.T.) 2 The Francis Crick Institute, London NW1 1AT, UK 3 Moorfields Eye Hospital NHS Foundation Trust, London EC1V 2PD, UK 4 Great Ormond Street Hospital for Children NHS Found Trust, London WC1N 3JH, UK * Correspondence: [email protected]; Tel.: +44-207-608-6971 Abstract: Inherited retinal diseases (IRDs) are a heterogeneous group of disorders causing progres- sive loss of vision, affecting approximately one in 1000 people worldwide. Gene augmentation therapy, which typically involves using adeno-associated viral vectors for delivery of healthy gene copies to affected tissues, has shown great promise as a strategy for the treatment of IRDs. How- ever, the use of viruses is associated with several limitations, including harmful immune responses, genome integration, and limited gene carrying capacity. Here, we review the advances in non-viral gene augmentation strategies, such as the use of plasmids with minimal bacterial backbones and scaffold/matrix attachment region (S/MAR) sequences, that have the capability to overcome these weaknesses by accommodating genes of any size and maintaining episomal transgene expression with a lower risk of eliciting an immune response. Low retinal transfection rates remain a limita- tion, but various strategies, including coupling the DNA with different types of chemical vehicles (nanoparticles) and the use of electrical methods such as iontophoresis and electrotransfection to aid Citation: Toualbi, L.; Toms, M.; Moosajee, M.
    [Show full text]