9 Gene Therapy in Correcting Birth Defects and Neurological Disorders MAYADHAR BARIK1*, M INU BAJPAI1, ARUN M ALHOTRA2, J.C. SAMANTARAY3 AND S.N. DWIEDI4
ABSTRACT
Gene therapy (GT) aims to fix a disease linked with genetic abnormality. In correcting birth defects, craniosynostosis and neurological disorders are currently receiving attention from the scientists, clinicians and the general public in an attempt to correct genetic defects including congenital abnormalities and neuro developmental disorders. However its application is beset with complications partly because of lack of advanced knowledge and research and partly because of vested interest by the society. This study is an attempt to highlight some of the major benefits of gene therapy and to define the role played by the medical informatics in facilitating rational use of gene therapy. This study is now attempting to review major studies in the international literature to synthesize the potential risks, benefits of GT. Which leads to define the role played by medical informatics in facilitating rational use of GT. An attempt was made to review major studies in the international literature to synthesize
1
Department of Pediatric Surgery, All India Institute of Medical Sciences, New Delhi-110029, India 2 Department of Nuclear Medicine, All India Institute of Medical Sciences, New Delhi-110029, India 3 De partme nt of Micro biolo gy, All India Institute of Medical Scie nces, New Delhi-110029, India 4 Department of Bio statistics, All India Institute of Medical Science s, N ew Delhi-110029, India *Corresponding author: E-mail:
[email protected] m, mayadharbarik@ rediffmail.com
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the potential risks, benefits of gene therapy. We also reviewed the recent advanced tools and techniques in the field of medical informatics. Key words: Gene therapy (GT), New gene therapy (NGT), Insertion gene therapy (IGT), Fetal hemoglobin (HbF), Human HGF plasmid DNA therapy (HHPDT) INTRODUCTION
Gene therapy (GT) has traditionally been used to refer to the concept of correcting genetic diseases at the genetic level. More recently, several studies have shown that molecular genetics techniques can be applied to non genetic as well as genetic diseases. As a result, the term gene therapy has taken on a broader definition, referring to the use of molecular genetics techniques in general for the treatment of diseases[4,5,6]. Chromosomes and Genes Every disease and human behavior is genetically determined and it is codified in the genes which are located in the nuclei and mitochondria of the cells. There are 23 pairs (total 46 chromosomes) of chromosome in each cells, 22 pairs of autosomes and each pair has identical or homologous genetic material. There is one pair of sex chromosomes which is homologous in females which have XX chromosomes with one X-chromosome and a much smaller Y-chromosome. This is according to chromosomal theory of T.H. Morgan and artificial gene developed by Dr. Harogovind Khurana. Chromosomal Disorders There may be loss or gain of a whole chromosome (monosomy, trisomy) or its part (translocation). Chromosomes have two arms which are designated as short (p) and long (q) arms. They are joined by a constriction known as centromere (c). Chromosomal abnormalities are generally sporadic (without any risk of recurrence) and occur in 0.5% of live born babies. The various mechanisms of production of chromosomal abnormalities are non-disjunction, mosaicism, translocation, addition and deletion. Common types of chromosomal disorders are trisomy-21 (down syndrome), trisomy-18 (edward syndrome), trisomy-13 (patau syndrome), turner syndrome 45X or 45 X/46 XX mosaic), klinefelter syndrome (47XXY or XXY/XY mosaic) and fragile X syndrome are disorder of sex chromosome.
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Genetic Disorders Most of the five types of genetic disorders are environmental, chromosomal, single-gene, polygenic, mitochondrial and geneticallycontrolled somatic cell disorders are creating many types of diseases. Because of radiation effect, spontaneous abortion, heavy metal toxicity and insufficiency of iron, folic acid also cause of genetic disorders. Inborn Errors or Metabolism (IEM) IEM are single gene disorders due to deficiency of one or more enzymes which lead to defective synthesis, metabolism, transport, or storage of biochemical compounds. The incidence of IEM is estimated to be about 5-6 per 1000 live births. IEMs are recognized mainly carbohydrate disorders, lipid disorder, amine acid disorders, primers and pyramiding disorders, parental consanguinity, develop and mental retardation. Research need to improve this area to know the reality. Prevention of Genetic Disorders (POGD) Preventive strategies should be effectively harnessed to prevent the bir th of affec ted children like carr ier screening, folic acid supplementation, prenatal diagnosis, non-invasive screening, invasive prenatal testing, newborn screening general management, specific therapy, replacement therapy, reducing accumulation of toxic metabolite, promoting excretion of toxins, induction of enzymes, avoidance of certain drugs and organ transplantation. POGD is highly an expensive and more experimental/technical part of receiver of specific therapy (ST) and replacement therapy (RT). Common Inborn Errors of Metabolism (CIEOM) CIEOM are normally phenylketonuria, tyrorinemia, albinism, alkaptonuria, homocysteininuria, hartnup disease, male syrup urine disease (MSUD), galactosemia, hereditary fructose intolerance, glycogen storage disorders, von gierk disease (Type 1GSD), pompe disease (type 2GSD), mucopoly saccharidosis, gaucher disease, neimann-pick disease, taysachs diseases, zellweger syndrome and alpha-1-Antfrypein deficiency. Gene or Gene Production Most recessive genetic diseases result from the loss of a specific genetic function, it is necessary to identify the mutant gene, clone its normal homologue, and introduce a functional copy into the diseased cells.
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Strategy for Gene Transfer Four general strategies have been developed for gene therapy: the in vivo approach, ex vivo, in vitro approach and recent advanced approach. Each of these four approaches have potential advantages and disadvantages that render them appropriate under different conditioning. According to characterization of diseases the molecules are to be transfer red in proper way. Molecular Technique for Inducing the Gene The simplest approach to the introduction of foreign genetic material into a patient might be the direct injection of DNA, RNA or nucleic acids into an affected area. This approach requires that naked genetic material be taken up by cells and expressed. Responsible vectors of gene therapy are retovirus, herpes simplex virus (HSV) and adenovirus. Cell Type and Region Neurologic diseases are, important to identify the type and location of the cell targeted for genetic manipulation. Resulting from a disturbance of ubiquitous metabolic processes, it may be necessary to introduce genetic material into all cells throughout the central nervous system (CNS). Genetic modification of glial cells or myocytes may be required for the correction of diseases of myelin and muscle, respectively. Materials and Methods It is always appreciated that international methods should be applied for this therapeutics to made success. In prior to retroviruses are a class of viruses treating as double-stranded DNA (ds DNA) copies of their RNA genomes. Selected copies of its genome to integrate into respective chromosomes host cells, is example of HIV. Adenoviruses are also a class of viruses with inserting their genetic material at a specific site of chromosome. Another way herpes simplex virus (HSV) is infecting a particular cell type, neurons, HSV type I is very common pathogen, which causes cold sores. Results The caused of following examples related to birth defects and numliogical disorders have been discussed in a section on discussion.
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Age of the Affected Host The age of the subject targeted for gene therapy is an important variable, since many diseases affecting the nervous system are associated with irreversible developmental or neurodegenerative processes. Another way if we observe any specific age affected host cells are responsible for other diseases. Gene Therapy in Cancer GT approach to brain tumors exploits differences in the growth characteristics of malignant cells and mature brain cells. Like transgenic introduced into cells by retroviral vectors require cell division for stable expression. Since many malignant cells divide rapidly whereas normal brain cells are quiescent, retroviral vectors can be used to deliver genes selectively to the malignant cells. Stem Cell and Gene Therapy in Molecular Medicine Several independent studies have reported the possibility of using genetically modified bone marrow cells in the treatment of lysomal storage disorders. Like galactoceremidase is a lysosomal enzyme responsible for the degradation of myelin glycoprotein. Galactoceramid are deficiency in krabbe’s disease is associated with a pathologic accumulation of cerebrorides and psycho sine and results in defective myelination in both the central and peripheral nervous system (PNS)[9,10]. Genetic Counseling Genetic counseling is a process to assist the parents and family to understand the nature of genetic disorder, its risk of transmission to subsequent siblings, available options for prenatal diagnosis and medical termination of pregnancy. In this explanation we have to use both (mendelian and non-mendelian principles). Gene Therapy in Medical Informatics GT with replacement of abnormal gene is curative but is still in experimental stage. GT has been tried in patients with adenosine deaminase deficiency (ADA) duchenne muscular dystrophy, familial hypercholesterolemia and certain cancers. The aim is to replace the deactive gene with the normal gene. This is done by using a viral or non-viral vector for introducing the normal functioning gene (NFG).
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DISCUSSION
In corroborating with viruses-mediated gene delivery systems are maximum non-viral. However, direct introduction of therapeutic DNA into target cells, is easier than others. Its application only with certain tissues requires larger amounts of DNA. Non-viral approach involves the creation of an artificial lipid sphere with an aquous core. We here mention that liposome carries the therapeutic DNA which is capable of transmitting the DNA through the specific target cells membrane. Where, therapeutic DNA getting inside target cells by chemically linking the DNA to a molecular level binds to special receptors. Which is bound to receptors, the therapeutic DNA constructs are engulfed by cell membrane and passed into interior target cell. The potential method is the difficulty in delivering these larger molecules to nucleus of a target cell. Many inherited metabolic diseases will not require complete restoration of gene function for correcting important aspects of the disease phenotype. E.g. in parkinsonism phenylketouria and other diseases. Pseudogene and transgenic expression is needful (examples are given in Table 1). In Table 1 we have highlighted inborn errors related diseases. In this condition autosomal recessive (AR), chromosomal translocation (CT) and familial cases should not be ignored. In general view of both the significance of the role for somatic gene therapy and molecular mechanisms for its implementation have evolved dramatically now days (examples are given in Table 2). In this table we noticed to researchers and clinicians that GT, NGT, copenrnicus therapeutics, hydroxyurea, fetal hemoglobin, HHPDT and IGT is more important because in this condition expertisation is more needed. Distinguished with another area of change is the expansion of the delivery system, which began retroviral vectors but now includes vectors based on adenovirus, adeno-associated viruses (AAV), herpes virus, vaccine, and other agents are documented non-viral systems(NVS) such as lipsomes, DNA-protein conjugates, and DNA-protein-defective virus conjugates are also promising. In a meanwhile retroviral vectors have the major advantages that they integrate with the foreign DNA and permanently alter the recipient cell, while they have two major disadvantages are the requirement for dividing cells such as a target and they have to provide low-titer virus, which is relatively impractical for most in in-vivo approaches. In-contrast to adenovirus vectors offer as high titer and better ability to effect large numbers of cells in-vivo, but there is concern about toxic effects on infected cells, and the therapeutic effects is only transient. Both in-vivo and in-vitro have been good. GT has so far been reported to be useful in tow genetic disorders, ADA deficiency and hypercholesterolemia due to defect in the LDL rec eptor protein. Many trials have been approved for many
Alkaptonuria Cystinuria Galactosaemia Gaucher’s diseases G-6-PD deficiency
Hunters syndrome
Isoniazidinactivations Niemann-Pick Disease Phenyl ketonuria Prophyria (Acuteintermittent)
Tay-Sachs diseases Vitamin-D Wilson’s diseases
Hurler’s syndrome Achondroplasia Cystic fibrosis
Chronic
Parkinson diseases
3. 4. 5. 6. 7.
8.
9. 10.
13. 14. 15.
16. 17. 18.
19.
20.
Synuclein protein
AR AD AR
-Iduronidase yrosine kinase Proteincystic fibrosistransmembrane conductor regulator mylogenusleukemia White blood cells
FM
CT
AR Renal AR
AR AR
AR AR
Hexosaminidase-A resistantRickets Not Known
Phenyl alaninehydroxylase More hepatic ALA synthestase
N-acetyl transferase Sphingomyelinase
Sulphoid duronatesulphatase
AR ARXLinked AR AR AR AR XLinked XR
Inheritance
Homogentisic acidoxidase Not Known Galactose-1-phosphate uridyl transferase Glucocerebrosidase Glucose-6-phosphate dehydrogenase
Catalase Tyrosinase
Enzyme
*AR- Autosomal recessive, CT-Chromosomal translocation, FM-Familial
11. 12.
Actalasia Albinism
Disorder
1. 2.
S. no.
Table 1: Inborn errors of metabolism important examples
Increased and unregulated growth of predominantly myeloid cells in the bone marrow. Tremor at rest, stiffness, slowing of movement and postural instability.
Haepatosplenomegaly, mentalretardation, skeletal abnormalities. Neurological Problems. CNS damaged, cherry red spot on macula and hepatospelanomegaly Microcephaly, mentalretardation Acute abdominal painepisode, neurological problems, excessive excreation of amino-levu-linic acid (ALA) in urine. Convulsions, Mentalretardation, Blindness defect inphosphate reabsorption XD Rickets Cirrhosis of liver, kayser-Flesher ring in cornea, neurological problems. Same as above, in addition there is corneal clouding. Skeletal Deformity Critically lungs, pancreasliver and intestine.
Arthritis Renal stores, aminoaciduria Mental retardation, contract, cirrhosis Hepatosplenomegaly, Thrombocytopenia and anaemia Haemolysis in response tosome drugs.
May present as oral gangrene. Lack of pigment in skin, hair and eyes.
Clinical manifestations
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Table 2: Major developments in Gene therapy (GT) S. no.
Diseases
Causes/Through
1. 2. 3.
Genetic defect Defective gene Thalassaemia
ADA-SCID M-RNA Blood disorder
4. 5. 6.
Cystic fibrosis Some cancers Sickle-cell diseases
– – HbF
7. 8.
Hereditary diseases Severe combined immune deficiencyor “bubble boy” diseases Parkinson diseases Huntington’s diseases Coronary artery diseases
Hematopoietic Stem Cells Hematopoietic Stem Cells
9. 10. 11.
Approach GT NGT Copen-rnicus therapeutics – – Hydroxyurea/ HbF GT GT
Polyethylene glycol IG RNA Interference/Genesilencing GT Cardiomyocytes HHPDT
*GT-Gene therapy, NGT-New gene therapy, IGT- Insertion gene therapy, HbF Fetalhemoglobin, HHPDT-Human HGF plasmid DNA Therapy
malignancies, AIDS, cystic fibrosis, gauchers diseases, a craniofacial defects (example given in Table 3). In Table 3 we want to establish the new gene therapy(NGT) and antisense oligonucleotides (AONS). Table 3: Most promising and gene therapy need to be improved according to our experience. S. no. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Diseases Di George Syndrome Velocardio facial Syndrome Vander Woude Syndrome Treacher Collins Syndrome Saethre-Chotzen Syndrome Cleidocranial Dysplasia Holoprosencephaly Adelaide Type Greig Syndrome Stickler Syndrome Waardeburg Syndrome
Chromosomal location
Approach
Di George: 22q NGT Velocardio facial: 22q NGT Vander Woude: 1q NGT Treacher Collins: 5q NGT Saethre-Chotzen: 7p NGT Cleidocranial Dysplasia: 6p NGT Holoprosencephaly: 2p, 7q NGT Adelaide Type CS: 4p NGT Greig: 7p, GLI-3 NGT Stickler: 12q, COL2A1, 6p, COL11A2 NGT Waardeburg I: 2q, PAX3, NGT 11:3p, MITF Boston Types CS Boston Types CS: 5q, MSX2 NGT Crouzon Syndrome Crouzon: 10q, FGFR2 NGT Pfeiffer Syndrome Pfeiffer: 8p, FGFR1, 10q, FGFR2 NGT Jackson-Weissman Syndrome Jackson-Weiss: 10q, FGFR2 NGT Achondroplasia Achondroplasia: 4p, FGFR3 NGT Thanatophoric Dysplasia Thanatophoric Dysplasia: 4p, NGT FGFR3 Duchenne’s Muscular DMD Gene: Exon-51 deletion Antisense Dystrophy oligonucleotide PROO51 Edward Syndrome Edward: trisomy-18 NGT Down Syndrome Down: trisomy-21 NGT
*NGT-New gene therapy.
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Tools and Techniques As per the development of recent advancement like microarray, next generation sequencing, SSCP, SNPs, DDGE, RFLP, blotting, karyotyping, ARMS, direct sequencing, VNTRS, microsatellites and other molecular techniques are more helpful to developed these therapeutics. The normal gene may be introduced directly into the defective body organ i.e. in-vivo or more often the process is achieved in-vitro followed by its introduction into the patient. According to availability of therapies have a major cost constraint. But very important and logistic issue is treatment modalities are new and emerging. It should be monitored and instituted at centers with sufficient experience and expertise. In the absences of definite treatment modalities it is more informative and accurate to improve all clinicians must familiar with counseling and preventive strategies to save many lives. Presently medical informatics well developed to give E-learning, simulation, animation, media played well role for betterment of treatment and future research. In the present scenario it seems that this new form of therapy is far from routine bed side clinical application. Being a skilled researcher, geneticist and scientist should focus on contamination and biosafety of laboratory as well as laboratory condition. It is an infancy that still single-gene disorders like syndromic and non-syndromic craniosynostosis diseases. Future of GT GT or molecular medicine still not well documented or progressed. Though it is highly promising and very serious threaten in near future. Researcher, scientist, clinicians, paramedic, tissue engineers should focus on these cases. Government and funding agencies should be taken necessary action for these infants (like FDI Clinical trials). Even suicide gene therapy is one of the most promising therapies to treat mesothelioma. We believe that virus, doctors and scientist introduce a protein-producing gene that converts a non-toxic drug into one that kill cancer cells. Cytokines are proteins that control and direct our immune response. It helps immune systems mount an attack against cancer cells. We agreed that so many challenges are facing GT like, controlled gene expression, getting genes to their proper targets, preventing destruction of the gene, delivery methods, condition of the host and host immune response should not be ignored. We were kin interest to develop and look after in future. At the same time viruses, viral delivery methods, Liposome delivery, and targeted gene delivery in cellular level in-vitro and in-vivo need to be improved.
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Side Effects GT and its potential complications the long term side effects in treatment remain unclear. Though it has not yet been observed through researchers are concerned that healthy cells may also become infected by the modified viruses, which could possibly causes new diseases or cancer to develop. For clearing this difficult concepts we need time to change and challenge. CONCLUSIONS
Before stopping my ink, GT techniques will be useful not only diagnosis of disease, but also treatment as well. REFERENCES [1]
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