1. Genetic disorders and Gene Therapy Course: Pharmaceutical Biotechnology (PHR- 403) Mir Ishruna Muniyat

2. What are Genetic Disorders? Genetic disorders are illnesses stemming from errors in a person’s genes Genetic disorders are illnesses stemming from errors in a person’s genes Any mistake in a gene can alter how a specific protein is produced Any mistake in a gene can alter how a specific protein is produced Without proper proteins, the body will not function properly and will take on a chronic and possibly life threatening condition Without proper proteins, the body will not function properly and will take on a chronic and possibly life threatening condition

3. How is it Caused? Genetic disorders can be congenital meaning they occur from birth or they may develop over time Genetic disorders can be congenital meaning they occur from birth or they may develop over time Congenital disorders occur when parents who are carriers of the disorder pass on their genes to their children Congenital disorders occur when parents who are carriers of the disorder pass on their genes to their children Progressive disorders that take time to set in may be caused by a mutation in DNA Progressive disorders that take time to set in may be caused by a mutation in DNA

4. Genetic Diseases Cystic Fibrosis Blood Disorders Muscular Dystrophy Diabetes

5. Acquired Diseases Cancer Cardiovascular Neurological Disorders Infectious Disease (AIDS)

6. What are recessive and dominant genes? Since human cells carry two copies of each chromosome, they have two versions of each gene. These different versions of a gene are called alleles. Since human cells carry two copies of each chromosome, they have two versions of each gene. These different versions of a gene are called alleles. Alleles can be wither dominant or recessive. Alleles can be wither dominant or recessive. Dominant alleles show their effect even if the individual only has one copy of the allele (also known as being heterozygous). Dominant alleles show their effect even if the individual only has one copy of the allele (also known as being heterozygous). For example, the allele for brown eyes is dominant, therefore you only need one copy of the 'brown eye' allele to have brown eyes (although, with two copies you will still have brown eyes). For example, the allele for brown eyes is dominant, therefore you only need one copy of the 'brown eye' allele to have brown eyes (although, with two copies you will still have brown eyes).

7. Recessive alleles only show their effect if the individual has two copies of the allele (also known as being homozygous). Recessive alleles only show their effect if the individual has two copies of the allele (also known as being homozygous). For example, the allele for blue eyes is recessive, therefore to have blue eyes you need to have two copies of the 'blue eye' allele. For example, the allele for blue eyes is recessive, therefore to have blue eyes you need to have two copies of the 'blue eye' allele. If both alleles are dominant, it is called codominance. The resulting characteristic is due to both alleles being expressed equally. If both alleles are dominant, it is called codominance. The resulting characteristic is due to both alleles being expressed equally. An example of this is the blood group AB which is the result of codominance of the A and B dominant alleles. An example of this is the blood group AB which is the result of codominance of the A and B dominant alleles. What are recessive and dominant genes?

10. What are sex-linked genes? Some genes are found on the sex chromosome, X. Some genes are found on the sex chromosome, X. These genes are inherited with the X chromosome (from the mother if it is a boy or from either mother or father if it is a girl). These genes are inherited with the X chromosome (from the mother if it is a boy or from either mother or father if it is a girl). Females have two X chromosomes (XX), while males have one X chromosome and one Y chromosome (XY). Females have two X chromosomes (XX), while males have one X chromosome and one Y chromosome (XY). This means females have two alleles for X-linked genes while males only have one. This means females have two alleles for X-linked genes while males only have one. Some genetic diseases, are caused by sex linked genes, for example haemophilia. Some genetic diseases, are caused by sex linked genes, for example haemophilia.

11. The allele for haemophilia is recessive so two copies are needed for a female to have the disease. The allele for haemophilia is recessive so two copies are needed for a female to have the disease. However, because males only have one X chromosome, they only need one copy of the haemophilia allele to have the disease. However, because males only have one X chromosome, they only need one copy of the haemophilia allele to have the disease. This means haemophilia is much more common in males than in females. This means haemophilia is much more common in males than in females. What are sex-linked genes?

12. Genetic disorders  Chromosomal disorders  Single Gene disorders Chromosomal disorders: Chromosomal disorders:  Gametes have abnormal chromosome numbers and mutations  Offspring inherit extra chromosome or are missing a chromosome  Caused by problems with meiosis

13. Genetic disorders Chromosomal disorders: Chromosomal disorders: Nondisjunction of chromosomes during meiosis Nondisjunction of chromosomes during meiosis

14. wide, rounded faceenlarged tongue Lower cognitive abilityequal length fingers webbed neck Chromosomal disorders: Example: Down Syndrome Down Syndrome Characteristics: Chromosomal disorders: Example: Down Syndrome Down Syndrome Characteristics: Normal female karyotype with 46 chromosomes Down syndrome karyotype with extra chromosome 21 Genetic disorders

15. Single gene disorders:   Caused by Mutations incorrect sequences in the normal form of the gene.   About 4,000 human diseases are thought to be inherited.   Scientists are making good progress figuring out where genes are located on chromosomes. Single gene disorders:   Caused by Mutations incorrect sequences in the normal form of the gene.   About 4,000 human diseases are thought to be inherited.   Scientists are making good progress figuring out where genes are located on chromosomes.

16. What is Gene Therapy? Gene therapy is the process of taking DNA coded for specific genes and adding it to a person’s genome in efforts to treat and alleviate genetic disorders Gene therapy is the process of taking DNA coded for specific genes and adding it to a person’s genome in efforts to treat and alleviate genetic disorders There are two types of treatment, In Vivo and Ex Vivo There are two types of treatment, In Vivo and Ex Vivo

17. Gene therapy Gene therapy: Gene therapy: to correct a genetic defect by transferring of a functional normal copy of the gene into cells to correct a genetic defect by transferring of a functional normal copy of the gene into cells There are four approaches: There are four approaches:  A normal gene inserted to compensate for a nonfunctional gene (Gene transplantation).  An abnormal gene traded for a normal gene (Gene transplantation)  An abnormal gene repaired through selective reverse mutation (Gene correction)  Change the regulation of gene pairs (Gene augmentation)

18. Examples of diseases caused by genetic defect Examples of diseases caused by genetic defect Ornithine transcarbamylase (OTC deficiency) Ornithine transcarbamylase (OTC deficiency) Hemophilia (blood coagulation factors VIII or IX) Hemophilia (blood coagulation factors VIII or IX) SCID( severe combined immunodeficiency) SCID( severe combined immunodeficiency) Muscular dystrophy Muscular dystrophy Cystic fibrosis Cystic fibrosis Sickle cell anemia Sickle cell anemia

19. Basic types of gene therapy The idea of gene therapy is a simple and logical one, fix a faulty gene by replacing it with a healthy gene. The idea of gene therapy is a simple and logical one, fix a faulty gene by replacing it with a healthy gene. However, there are different approaches scientists take in order to do so. However, there are different approaches scientists take in order to do so. The Different Types of Gene therapy include: The Different Types of Gene therapy include: 1) Germ Line Gene Therapy 1) Germ Line Gene Therapy 2)Somatic Cell Gene Therapy 2)Somatic Cell Gene Therapy 3) Chimeraplasty 3) Chimeraplasty

20. 1) Germ Line Gene Therapy This process involves the altering of a baby's the genome before it has even been born. This process involves the altering of a baby's the genome before it has even been born. It is still an emerging technique that needs to be perfected before being tested on humans. It is still an emerging technique that needs to be perfected before being tested on humans. Germ line therapy is also, a more challenging than the more common somatic cell gene therapy. Germ line therapy is also, a more challenging than the more common somatic cell gene therapy. However, germ line therapy raises concerns regarding ethics and morality. However, germ line therapy raises concerns regarding ethics and morality.

21. The two main methods of performing germ-line gene therapy would be: To treat a pre-embryo that carries a serious genetic defect before implantation in the mother, with the use of in-vitro fertilization The two main methods of performing germ-line gene therapy would be: To treat a pre-embryo that carries a serious genetic defect before implantation in the mother, with the use of in-vitro fertilization To treat the germ cells (sperm or egg cells) of afflicted adults so that their genetic defects would not be passed on to offspring approach to delete the defective gene and insert a properly functioning replacement. To treat the germ cells (sperm or egg cells) of afflicted adults so that their genetic defects would not be passed on to offspring approach to delete the defective gene and insert a properly functioning replacement. 1) Germ Line Gene Therapy

22. 2) Somatic Cell Gene Therapy The most studied gene therapy, somatic cell therapy uses the insertion of a normal gene into the DNA of somatic cells in order to compensate for the non-functioning defective gene. The most studied gene therapy, somatic cell therapy uses the insertion of a normal gene into the DNA of somatic cells in order to compensate for the non-functioning defective gene. This can be done in a number of ways including: This can be done in a number of ways including: Inserting the gene into any location within the genome to replace a nonfunctional gene, which is the most commonly used Inserting the gene into any location within the genome to replace a nonfunctional gene, which is the most commonly used Switching the abnormal gene for a normal gene through homologous recombination. Switching the abnormal gene for a normal gene through homologous recombination. Fixing through selective reverse mutation, which returns the gene to its normal function. Fixing through selective reverse mutation, which returns the gene to its normal function. Somatic Gene therapy delivers the normal gene into a somatic or body cell of an individual. This allows for specific locations of the body to be targeted but also limits the inheritance of the correct genome to future generations. Somatic Gene therapy delivers the normal gene into a somatic or body cell of an individual. This allows for specific locations of the body to be targeted but also limits the inheritance of the correct genome to future generations.

23. 3) Chimeraplasty This technique is the least known of all three methods. It is a non- viral method that is still being researched for its potential in gene therapy. This technique is the least known of all three methods. It is a non- viral method that is still being researched for its potential in gene therapy. Chimeraplasty is done by changing DNA sequences in a person's genome using a synthetic strand composed of RNA and DNA. Chimeraplasty is done by changing DNA sequences in a person's genome using a synthetic strand composed of RNA and DNA. This strand of RNA and DNA is known as a chimeraplast. The chimeraplast enters a cell and attaches itself to the target gene. This strand of RNA and DNA is known as a chimeraplast. The chimeraplast enters a cell and attaches itself to the target gene. The DNA of the chimeraplast and the cell complement each other except in the middle of the strand, where the chimeraplast's sequence is different from that of the cell. The DNA repair enzymes then replace the cells DNA with that of the chimeraplast. This leaves the chimeraplast's new sequence in the cell's DNA and the replaced DNA sequence then decays. The DNA of the chimeraplast and the cell complement each other except in the middle of the strand, where the chimeraplast's sequence is different from that of the cell. The DNA repair enzymes then replace the cells DNA with that of the chimeraplast. This leaves the chimeraplast's new sequence in the cell's DNA and the replaced DNA sequence then decays.

24. Factors to be considered in Gene therapy How to deliver genes to specific cells, tissue and whole animals? (methods of delivery) How to deliver genes to specific cells, tissue and whole animals? (methods of delivery) How much and how long the introduced gene will be expressed? How much and how long the introduced gene will be expressed? The site and dose of gene delivery The site and dose of gene delivery Is there any adverse immunological consequence of both delivery vehicle (Virus) and the gene in animals? Is there any adverse immunological consequence of both delivery vehicle (Virus) and the gene in animals? Is there any toxic effects? Is there any toxic effects?

25. Methods of gene delivery Viral Vectors: Viral Vectors: Adenovirus Adenovirus Retrovirus Retrovirus Lentivirus Lentivirus Adeno-associated virus (AAV) Adeno-associated virus (AAV) Herpes simplex virus (HSV) Herpes simplex virus (HSV) Non-viral vector based Non-viral vector based Naked DNA (plasmid DNA): injection or genegun Naked DNA (plasmid DNA): injection or genegun Liposomes (cationic lipids): mix with genes Liposomes (cationic lipids): mix with genes Ex-vivo Ex-vivo In vivo In vivo

26. Methods of gene delivery There are also various ways in which target cells can be isolated for integration. There are also various ways in which target cells can be isolated for integration. Regardless of the type, each therapy requires a vector to transport DNA into cells Regardless of the type, each therapy requires a vector to transport DNA into cells Ex vivo requires a removal of target cells from the patient, growing them in a culture, and introducing the vector into the culture. Ex vivo requires a removal of target cells from the patient, growing them in a culture, and introducing the vector into the culture. After the target cells have integrated the DNA, they are replaced into the patient. After the target cells have integrated the DNA, they are replaced into the patient. In vivo treatment simply requires the injection of the vector that is specific to target cells into the patient. In vivo treatment simply requires the injection of the vector that is specific to target cells into the patient.

27. Since viral vectors are commonly regarded as the most effective delivery systems, many methods have been researched and utilize various types of viruses: retroviruses, adenoviruses, adeno-associated viruses, and the herpes simplex virus are common. Since viral vectors are commonly regarded as the most effective delivery systems, many methods have been researched and utilize various types of viruses: retroviruses, adenoviruses, adeno-associated viruses, and the herpes simplex virus are common. These viruses differ in how well they transfer genes to the cells they recognize and are able to infect, and whether they alter the cell’s DNA permanently or temporarily These viruses differ in how well they transfer genes to the cells they recognize and are able to infect, and whether they alter the cell’s DNA permanently or temporarily Methods of gene delivery

30. Vectors of Gene Therapy “Vectors" refer to the method of gene insertion. “Vectors" refer to the method of gene insertion. Non-viral vectors Non-viral vectors plasmids cultivated in bacteria and naked plasmids that are inserted directly into the cell. plasmids cultivated in bacteria and naked plasmids that are inserted directly into the cell. Non-viral vectors are not cell specific but are generally less effective in their integration into the host cell. Non-viral vectors are not cell specific but are generally less effective in their integration into the host cell. Viral vectors Viral vectors Refer to modified viruses used to deliver DNA into target cells. Refer to modified viruses used to deliver DNA into target cells. Viral vectors are usually more specific to certain cell types but are limited in the size of the genome they can fit inside their capsid. Viral vectors are usually more specific to certain cell types but are limited in the size of the genome they can fit inside their capsid. Viral vectors may cause an immune response in the patient, or if the patient is inherently immune to the specific type of virus, then the success of DNA integration is diminished. Viral vectors may cause an immune response in the patient, or if the patient is inherently immune to the specific type of virus, then the success of DNA integration is diminished.

31. Non viral gene delivery systems Direct introduction of therapeutic DNA:

32. Non viral gene delivery systems Current attempts with naked DNA vaccination in infectious diseases:  HIV  Hepatitis  Influenza  Tuberculosis  Lyme disease  Malaria Current attempts with naked DNA vaccination in infectious diseases:  HIV  Hepatitis  Influenza  Tuberculosis  Lyme disease  Malaria

33. Non viral gene delivery systems Direct introduction therapeutic DNA: Ballistic DNA injections: Direct introduction therapeutic DNA: Ballistic DNA injections: Invented for gene transfer in plant cells Fully applicable to eukayotic cells Invented for gene transfer in plant cells Fully applicable to eukayotic cells plasmid DNA shown here

34. Therapeutic DNA Non viral gene delivery systems Gene delivery using Liposomes: Next level idea – why naked DNA? Lets’ wrap it in something safe to increase transfection rate Lets’ wrap it in something safe to increase transfection rate Lipids – are an obvious idea !

35. Non viral gene delivery systems Cheaper than viruses No immune response Especially good for in-lung delivery (cystic fibrosis) 100-1000 times more plasmid DNA needed for the same transfer efficiency as for viral vector Gene delivery using Liposomes:

36. Why use viral vectors Virus are obligate intracellular parasites (cannot reproduce outside their host cell) Virus are obligate intracellular parasites (cannot reproduce outside their host cell) Very efficient at transferring viral DNA into host cells Very efficient at transferring viral DNA into host cells Specific target cells: depending on the viral attachment proteins (capsid or glycoproteins) Specific target cells: depending on the viral attachment proteins (capsid or glycoproteins) Gene replacement: non-essential genes of virus are deleted and exogenous genes are inserted Gene replacement: non-essential genes of virus are deleted and exogenous genes are inserted Viruses can also be modified to express fewer capsid proteins and to express certain glycoproteins that are more specific to target cells. Viruses can also be modified to express fewer capsid proteins and to express certain glycoproteins that are more specific to target cells.

37. 1. Retrovirus vector Retroviruses are characterized by their use of the host cell's reverse transcriptase to create DNA from viral RNA. Retroviruses are characterized by their use of the host cell's reverse transcriptase to create DNA from viral RNA. The DNA is then incorporated into the host's DNA genome using integrase. The DNA is then incorporated into the host's DNA genome using integrase. integrase inserts the gene anywhere because it has no specific site integrase inserts the gene anywhere because it has no specific site May cause insertional mutagenesis (one gene disrupts another gene’s code. disrupted cell division causes cancer from uncontrolled cell division ) May cause insertional mutagenesis (one gene disrupts another gene’s code. disrupted cell division causes cancer from uncontrolled cell division ) Vectors used are derived from the human immunodeficiency virus (HIV) and are being evaluated for safety Vectors used are derived from the human immunodeficiency virus (HIV) and are being evaluated for safety Viral gene delivery systems

38. Characteristics of retroviral vector Advantages Advantages Integration: permanent expression Integration: permanent expression Pseudotyped virus Pseudotyped virus Disadvantages Disadvantages Only infecting dividing cells Only infecting dividing cells Insertional mutagenesis (tumor formation) Insertional mutagenesis (tumor formation) Activate oncogenes Activate oncogenes Inhibit tumor suppressor genes Inhibit tumor suppressor genes Note: * Pseudotyping is the process of producing viruses or viral vectors in combination with foreign viral envelope proteins. The result is a pseudotyped virus particle. With this method, the foreign viral envelope proteins can be used to alter host tropism or an increased/decreased stability of the virus particles.

39. 2) Adeno Associated virus  Small, single stranded DNA that insert genetic material at a specific point on chromosome 19  From parvovirus family- causes no known disease and doesn't trigger patient immune response.  Low information capacity  gene is always "on" so the protein is always being expressed, possibly even in instances when it isn't needed.  For example, for Hemophilia treatment,  a gene-carrying vector could be injected into a muscle, prompting the muscle cells to produce Factor IX and thus prevent bleeding.  Study by Wilson and Kathy High (University of Pennsylvania), patients have not needed Factor IX injections for more than a year

40. 3) Herpes Simplex virus  Double stranded DNA viruses that infect neurons  Ex. Herpes simplex virus type 1 Linear ds DNA, 152 kb, about half of the total 81 genes are non-essential for virus replication Linear ds DNA, 152 kb, about half of the total 81 genes are non-essential for virus replication 40-50 kb of foreign DNA can be accommodated 40-50 kb of foreign DNA can be accommodated Neurotropic virus, target to nervous system Neurotropic virus, target to nervous system

41. Adenoviral vectors Non-enveloped ds DNA, 36 kilobases Non-enveloped ds DNA, 36 kilobases Usually causes a benign respiratory infections in human Usually causes a benign respiratory infections in human Serotypes 2 and 5 are commonly used as vectors Serotypes 2 and 5 are commonly used as vectors

43. Death of 18-year old Jesse Gelsinger Liver disease: OTC deficiency (genetic disease) University of Pennsylvania High dose of adenoviral vector (E1 and E4 genes deleted ) carrying the normal copy of OTC gene was administered Suspected cause of death Toxicity of high titer adenoviral vector High immunogenicity of adenoviral vector (an immune revolt)

44. Characteristics of adenoviral vector Advantages Advantages High titers High titers Both dividing and non-dividing cells Both dividing and non-dividing cells Wide tissue tropism Wide tissue tropism Easily modify tissue tropism Easily modify tissue tropism Disadvantages Disadvantages Transient expression ( not good for genetic diseases) Transient expression ( not good for genetic diseases) Highly immunogenic Highly immunogenic High titers of virus can be toxic High titers of virus can be toxic More suitable for cancer immunotherapy More suitable for cancer immunotherapy

45. Lentiviral vectors Infection of non-dividing cells (hepatocytes, neurons) Infection of non-dividing cells (hepatocytes, neurons) HIV, a human lethal pathogen HIV, a human lethal pathogen Delete accessory genes Delete accessory genes Provide an envelope from a non-retrovirus (VSV) Provide an envelope from a non-retrovirus (VSV) Develop vectors from lentiviruses of non-human pathogens Develop vectors from lentiviruses of non-human pathogens SIV, FIV, EIAV etc SIV, FIV, EIAV etc

46. Adeno-associated virus vectors Non-pathogenic human parvovirus, non- enveloped ss DNA virus, 4.6 kilobases Non-pathogenic human parvovirus, non- enveloped ss DNA virus, 4.6 kilobases Dependent on a helper virus ( adenovirus or herpesvirus) for replication (dependovirus) Dependent on a helper virus ( adenovirus or herpesvirus) for replication (dependovirus) AAV-2 mostly used for vector AAV-2 mostly used for vector

47. Characteristics of AAV vector Advantages Advantages Integration and persistent expression Integration and persistent expression No insertional mutagenesis No insertional mutagenesis Infecting dividing and nondividing cells Infecting dividing and nondividing cells Safe Safe Disadvantages Disadvantages Size limitation, 4.9 kb Size limitation, 4.9 kb Low titer of virus, low level of gene expression Low titer of virus, low level of gene expression

48. Comparison of different viral vectors Viral vectortitersmanupilation of immunogenicityinfecting of tropism non-dividing cells Adenovirus10 11 terrificvery highyes Retrovirus10 7 goodlowonly lentivirus Herpesvirus10 7 not so goodlowyes AAV10 7 not so goodlowyes

50. Problems with gene therapy  Short Lived  Hard to rapidly integrate therapeutic DNA into genome and rapidly dividing nature of cells prevent gene therapy from long time  Would have to have multiple rounds of therapy  Immune Response  new things introduced leads to immune response  increased response when a repeat offender enters  Viral Vectors  patient could have toxic, immune, inflammatory response  also may cause disease once inside  Multigene Disorders  Heart disease, high blood pressure, Alzheimer’s, arthritis and diabetes are hard to treat because you need to introduce more than one gene  May induce a tumor if integrated in a tumor suppressor gene because insertional mutagenesis