Gene Therapy and Recombinant DNA

• Gene therapy is used to correct defective genes responsible for disease development
• Gene therapy has potential in treating disorders from a single gene defect
• Somatic gene therapy is not hereditary
• Germline gene therapy is hereditary, but illegal

Recombinant DNA

• Recombinant DNA is introduced into E. coli, where plasmid and bacteria replication occurs

Joining DNA Molecules

• Complementary base pairing occurs between insert DNA and vector DNA
• DNA ligase joins the DNA molecules

Gene Function in Eukaryotes

• Cloned DNA is introduced into plant and animal cells through gene transfer
• Transfection is the methodology used for infectious viral DNAs

Gene Transfection Methods:

• Viral transfection, such as with lentivirus can be used to transfect cells
• Direct microinjection into the nucleus
• Coprecipitation of DNA with calcium phosphate is used to form small particles to be taken up by cells
• DNA is incorporated into liposomes that fuse with the plasma membrane
• Electroporation involves exposing cells to a brief electric pulse that opens pores in the plasma membrane

Retroviral Vectors

• Retroviral vectors can deliver genes to mammalian cells via transfection
• A few cells then take up DNA and produce recombinant virus particles
• Recombinant retrovirus particles carry inserted DNA
• In new cells, efficient infection and gene expression can then occur
• Reverse transcription occurs in this process

Retrovirus Gene Therapy

• The process involves inserting the RNA version of a normal allele into a retrovirus
• The retrovirus infects bone marrow cells removed from the patient and cultured
• Viral DNA carrying the normal allele inserts into the chromosome
• Engineered cells inject back into the patient

Protein vs Gene Therapy:

• Type I diabetes: Insulin
• Pituitary dwarfism: GH
• Hemophilia: Factor VIII, IX
• Anemia: Erythropoietin

SCID (Severe Combined Immunodeficiency):

• SCID is inherited as an X-linked recessive disease
• In some patients, it presents as an autosomal recessive disorder
• First gene therapy on September 14, 1990: Ashanti DeSilva received treatment for SCID by having modified white blood cells injected back into her blood stream
• Her immune system was strengthened as a result

SCID Details

• There is an ADA gene defect (Adenosine deaminase)
• The immune system is weak in SCID patients
• ADA converts adenosine to inosine via deaminase (A → I)
• Adenosine and deoxyadenosine accumulation is toxic for lymphoid cells, mainly T-cells
• Untreated individuals without T-cells are prone to viral and bacterial infections
• Those patients are also called "bubble boy disease"
• SCID is associated with a mutation on Chromosome 20
• SCID patients struggle to gain weight, grow, and are prone to pneumonia and ear infections

SCID Gene Therapy

• PEG-ADA injections (synthetic ADA) provide treatment; resistance to the drug develops
• Bone marrow transplants are challenging without a donor
• Gene therapy involves collecting a patient's T-cells
• T-cells are then transfected with a ADA gene-containing retrovirus and injected back into the patient
• The date used for initial injections was September 14th, 1990

Gene Therapy Methods

• In ex vivo gene therapy, cells are manipulated outside the body before being injected back in
• In situ gene therapy involves injecting a healthy gene or vector DNA directly into an accessible area like a skin tumor
• In vivo gene therapy includes injecting gene or vector DNA directly into the body
• In vivo is delivery of genes taking place in the body
• Ex vivo is delivery of genes taking place outside of the body, and cells being placed back

In vivo Gene Therapy:

• Introduces genetic material directly into the patient's body
• Can be a random process with limited control and manipulation
• Only option when tissues cannot be grown in vitro or transferred back effectively

Ex vivo Gene Therapy:

• Transfers genetic material into cells grown in vitro
• It's a controlled process, with genetically altered cells selected and expanded
• The cells are then returned to the patient

Human Gene Therapy

• Vectors deliver genes to specific cell types, such as bone marrow
• Ethical issues arise regarding treating human germ-line cells to correct defects for future generations

Vectors in Gene Therapy:

• Viral vectors: Adenovirus, Retrovirus, Herpes virus
• Non-viral vectors: Electroporation using short pulses of high voltage, gene gun delivering gene coated in gold nono-particles, sonoporation delivering DNA into cell by ultrasound

Gene Therapy and Diseases:

• Hemophilia: Viral and non-viral vectors directly injected
• Familial Hypercholesterolemia: LDLR gene injected via ex-vivo or in-vivo
• Cancer: Metastatic melanoma may use adaptive immunotherapy with tumor-infiltrating lymphocytes and an IL-2 encoding vector
• In-vivo/ex-vivo gene therapy uses viral/non-viral vectors encoding tumor suppressor genes, cytokines, and alloantigens

Lymphocytes

• Under normal conditions, lymphocytes recognize and target tumor cells secreting proteins, but sometimes they don't
• Tumor-infiltrating lymphocytes extracted from tumors are modified with IL-2 inserted and injected back
• The modified cells go to tumors and secrete IL-2, recruiting more lymphocytes

Challenges in Gene Therapy:

• Delivery: Needs to be target-specific
• Expression: Sustained expression is needed
• Regulation: Must be regulated within the body
• Vector: Safe and efficient; should carry genes, target specific cells, avoid immune response and mutation, easy to produce, and have enough protein

Problems with Gene Therapy:

• May cause cancer
• Retroviral vectors in SCID cases can activate oncogenes, potentially resulting in leukemia
• High levels of adenovirus are good to kill cancer cells, but not good for regular treatment

Positive Developments:

• Anti-cancer drugs
• Recombinant human adenovirus for expressing p53
• Ocular gene therapy
• Arthritis
• Parkinson's
• Cystic Fibrosis
• Blindness

Successful Gene Therapy:

• Rhys Evans was born with SCID-X1
• His immune system was permanently repaired using gene therapy

Transgenic Animals

• Cloned genes are introduced into the germ line of multicellular organisms
• Microinjection of cloned DNA into a fertilized egg's pronucleus creates foreign gene-carrying mice (transgenic mice)

Embryonic Stem Cells

• Embryonic stem (ES) cells are used to introduce cloned genes into mice
• Cloned DNA is then introduced into ES cells in culture
• Stably transformed cells are introduced back into mouse embryos

In vitro Mutagenesis

• A common method of in vitro mutagenesis uses synthetic oligonucleotides to generate changes in a DNA sequence
• In vitro mutagenesis enables detailed characterization of the functions of regulatory and protein-coding sequences of cloned genes

Gene Inactivation

• Genes are inactivated in mouse embryonic stem cells, growing transgenic mice
• Mice are bred to yield progeny with mutated copies of the gene on homologous chromosomes
• The effects of inactivation are investigated within the animal
• Around 20% of mouse genes have been knocked out by homologous recombination; there is a global effort to knockout all mouse genes

Antisense Nucleic Acids

• Used to interfere with gene expression or function
• Consist of RNA or single-stranded DNA complementary to mRNA of the gene of interest
• They block the translation of mRNA into protein by hybridizing with the mRNA

RNAi Interference

• Double-stranded RNA degrades the target mRNA
• Double-stranded RNAs when introduced, are cleaved into short interfering RNAs (siRNAs) by the Dicer enzyme
• The siRNAs associate with the RNA-induced silencing complex (RISC), where mRNA is cleaved

Cloning Technique

• Egg cell from the cultured mammary cell undergoes nuclear transfer
• Early embryo is implanted into a surrogate mother
• Offspring are genetically identical to the mammary cell donor