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

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

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

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

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

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

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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")

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

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

The ssRNA (type V) Virus Life Cycle 1 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

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

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

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?

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.

PERAN REVERSE TRANSCRIPTASE Figure 5-72 Molecular Biology of the Cell (© Garland Science 2008)

HIV is a Retrovirus T-Cell Sadava figure 13.6

Human Retroviruses Are Used As Gene Therapy Vectors

Animal Viruses Are Used To Deliver Genes For Gene Therapy

The Retrovirus Life Cycle 1 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

The Retrovirus Life Cycle 1 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

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

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

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

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

HERPES SIMPLEKS

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)

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

Human Artificial Chromosome 47th chromosome Microchromosome, has only 6- 10 Mb size Developed after bacterial and yeast artificial chromosome Grown in HT1080 cell

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