1. Agustina Setiawati, M.Sc., Apt
GENE THERAPY
VEKTOR
Agustina Setiawati, M.Sc., Apt

2. VECTOR An adequate carrying capacity
To be undetectable by immune system
Safe to patient
High efficiency

4. Ex vivo manipulation techniques
Electroporation
Liposomes
Gold bullets (fired within helium pressurized gun)
Retrotransposons (jumping genes – early days)

6. In vivo techniques usually utilize viral vectors
Virus = carrier of desired gene
Virus is usually “crippled” to disable its ability to cause disease
Viral methods have proved to be the most efficient to date
Many viral vectors can stable integrate the desired gene into the target cell’s genome

7. VIRUS as VECTOR Retrovirus Adenovirus Adeno-associated virus
Herpes simplex virus

8. Comparison of Virus and Cell Sizes
Note: 1 nm = 10-9 m

9. 11

10. Viral genomes DNA viruses RNA viruses RNA  DNA viruses ss RNA ds DNA
(Retroviruses)
ds DNA
(hepadnaviruses)
ss DNA
ds DNA
ss RNA
ss RNA
genome can function as mRNA
genome is template for mRNA
genome is template for DNA synthesis ("retrovirus")

11. The (dsDNA) Virus Life Cycle
Protein
capsid
DNA 1
Virus enters host cell (method is variable, involves host receptor molecule on cell surface)
Viral DNA replicated using the host's DNA polymerase, nucleotides, etc.
DNA transcribed into mRNA using host's RNA polymerase, nucleotides
mRNA translated using host's ribosomes, tRNAs, amino acids, GTP, etc. 2 3
mRNA
DNA 4
capsid
proteins

12. The dsDNA Virus Life Cycle
Protein
capsid
The dsDNA Virus Life Cycle
DNA 1
New DNA and capsid proteins assemble into new virus particles, exit the cell (in various ways) 2 3
mRNA
DNA 4 5
capsid
proteins

13. The ssRNA (type V) Virus Life Cycle1
Virus enters host cell
Capsid removed, RNA released
complementary DNA made from genomic RNA by enzyme encoded in viral genome
new genomic DNA made from complementary strand
complementary strand is mRNA, transcribed into viral proteins
Virus assembled, exits cell (by various means) 2
RNA 3
cDNA 4
RNA 5 6

14. VIRAL LIFE CYCLE ATTACHMENT PENETRATION HOST FUNCTIONS UNCOATING
Transcription
Translation
REPLICATION
VIRAL LIFE CYCLE
ASSEMBLY
(MATURATION)
RELEASE
MULTIPLICATION

15. RETROVIRUS
RNA virus, so it need reverse transcription and integration when enter to the cell
Only divide in rapid proliferation cell (hair, intestine )
Insert their material genetic in the middle of important gene
Response is reduced by immune response

18. Retrovirus Life Cycle
Retroviruses Replicate Using Reverse Transcriptase
David Baltimore & Howard Temin-Nobel Prize 1975
Modified the Central Dogma of Molecular Biology
Use For Genetic Engineering?

19. group-specific antigen (gag) codes for core and structural proteins of the virus
polymerase (pol) codes for reverse transcriptase, protease and integrase
envelope (env) codes for the retroviral coat proteins.

20. PERAN REVERSE TRANSCRIPTASE
Figure Molecular Biology of the Cell (© Garland Science 2008)

21. HIV is a Retrovirus
T-Cell
Sadava figure 13.6

22. Human Retroviruses Are Used As Gene Therapy Vectors

23. Animal Viruses Are Used To Deliver Genes For Gene Therapy

24. The Retrovirus Life Cycle1
Virus enters host cell
Reverse transcriptase (encoded in viral genome) catalyzes synthesis of DNA complementary to the viral RNA (cDNA)
RTase catalyzes synthesis of 2nd strand of DNA complementary to the first
dsDNA incorporated into host genome ("provirus")
provirus may remain unexpressed for a period of latency
RTase
RNA 2
cDNA 3
Host's DNA 4 5 6

25. The Retrovirus Life Cycle1
Proviral genes are transcribed by host's transcriptional machinery into RNA
RNA serves as mRNA for translation into viral proteins and as genomic RNA
New viruses are assembled containing genomic RNA and Reverse Transcriptase
Virus exits cell
RTase
RNA 2
cDNA 3
Host's DNA 4 5 6

26. Using a Retrovirus as a Vector For Human Ex Vivo Gene Therapy
Endocytosis
Pol
Gag
Env
Grab from article
Pol = Reverse Transcriptase
Gag = Capid Protein
Env = Envelope Protein
 = Packaging Sequence

27. Using Retroviruses for Ex Vivo Gene Therapy
Cloning in Bacteria
DNA Transformation into Packaging Cell A.
Packaging Cells Makes Viral Proteins
Cannot Package (-Minus)
Packages Therapeutic Transcript (-Plus)
Packaging Cell Line
(Made Previously) B.
Grab from article C.
Infect Target Cells
Check For Presence of Gene
Transfer To Patient

28. ADENOVIRUS
DNA virus
Can infect slow proliferating cell, ie: blood, lung, skin
Material genetic can not be integrated to host DNA
Response is reduced by immune response

31. ADENO-ASSOCIATE VIRUS (AAV)
Occur naturally in human body
DNA virus
Harmless, non pathogenic, do not cause immune response
Insert their material genetic into chromosome 19 (spesific site)
Only 2 genes can be inserted to these vector
Trial to treat muscle and eye disease

33. HERPES SIMPLEKS

34. Non VIRAL VECTOR Artificial liposome
Human artificial chromosomes—introducing 47th chromosome to the cell
Chemical linking therapeutic DNA that can bind to specific receptor of cell
Cationic Liposome
Naked DNA (electroporation, gene gun, DNA injection)

35. Cationik liposome forms complex with DNA (negative charge)
Net charge of the complexes is positive so it can be interact to cell membrane

39. Human Artificial Chromosome
47th chromosome
Microchromosome, has only Mb size
Developed after bacterial and yeast artificial chromosome
Grown in HT1080 cell

41. Any question?

42. THANK YOU