Gene Delivery and Therapy Overview

Gene Delivery

• Gene delivery uses vectors to deliver genes into cells.
• Viral vectors and non-viral vectors are used.
• Viral vectors are effective at high protein production but have disadvantages like immunogenicity, mutation, and size limitation.
• Non-viral vectors are generally safe, and effective in delivering large amounts of transgene but have a lower level of protein production and transient expression compared to viral vectors.
• Retroviruses are RNA viruses that insert their genes permanently into host cell chromosomes after infection.

Gene Therapy

• Somatic gene therapy inserts genes into specific somatic cells to correct genetic diseases.
• Germ line therapy inserts genes into germ cells (e.g., fertilized eggs) which can be passed onto future generations.

Plasmids

• Plasmids are extra-chromosomal genetic elements that are circular and have double-stranded DNA molecules, with the capacity for autonomous replication in cells.
• Chromosomes contain essential genes for growth; plasmids have optional genes that confer additional traits. Cells can still function without lost plasmids.

Vectors for Gene Delivery

• Vectors are essential because genes cannot enter cells on their own.
• DNA is degraded in the body.

Retroviruses

• Retroviruses are RNA viruses that insert their genes permanently into host cell chromosomes after infection.
• To express a gene, retroviruses must reverse transcribe RNA to double-stranded DNA that is integrated into the host cell DNA.
• Retroviruses infect only dividing cells, facilitating ex vivo gene insertion, where cell division can be stimulated.

Viral Vectors (in Gene Therapy)

• Advantages of Viral vectors include high protein production
• Disadvantages include immunogenicity, mutation issues, size and limitation of transgene.

Retroviral Vector Safety Concerns

• Retroviral vectors can be directly administered to patients but their applicability is limited by rapid inactivation by human complement.
• Retroviral vectors are not safe, due to random insertion into host cell chromosome, which may lead to insertional mutagenesis and oncogenesis.

Viral Genome Components

• Viral genome components include genetic arrangement, gag (core proteins), pol (reverse transcriptase), env (viral protein coat), LTRs (long term repeat sequences that include promoter/enhancer regions), and packaging sequences (Ψ or Ø).

Retroviral Construction for Gene Delivery

• This process involves the following steps: -Therapeutic DNA replication -Transfection -Translocation (nucleus) and Therapeutic DNA Integration -Retroviral Genomic RNA Transcription -Packaging Cell Line for Virus Replication and Packaging steps -Retrovirus assembly -Recombinant Retroviruses Purification and Characterization -Amplification of Stocks for Administration

Retroviral Mediated Gene Delivery

• Retroviral vector internalized via receptor-mediated endocytosis.
• Retroviral vector is released from endosome, RNA is converted to DNA,
• Transported to the nucleus and randomly integrated into the host cell genome.
• Therapeutic DNA is expressed as a functional protein and inserted into the patient's genome after cell division.

Adenovirus Entry

• Adenovirus entry to the cell is receptor mediated with integrins, and CAR (coxsackie-adenovirus receptor).

Adenoviral Vector Construction

• Cloning of Therapeutic DNA
• Co-transfection with Replication-deficient Adenoviral Genome
• Homologous Recombination
• Translocation
• Replication
• Release of Adenovirus Particles
• Purification and Characterization of Recombinant Viruses
• Virus Amplification for Administration

Adenoviral-mediated Gene Delivery

• Adenoviral vector internalized via receptor-mediated endocytosis.
• Released from endosome and Translocated to the nucleus.
• Therapeutic DNA is transcribed and translated from episomal adenoviral genome.

Gene Therapy Success Stories

• Ashanti DeSilva, treated for SCID (1990). WBCs were collected and infused with the missing gene, then put back in, strengthening her immune system.
• NIH scientists treated metastatic melanoma in two patients (2006).
• Gero Hutter treated a man with HIV using gene therapy (2011).

Risk with Viral Vectors

• 1999: A patient with OTC deficiency who was treated with an adenovirus vector to deliver the OTC gene developed a high fever and died from multi-organ failure four days later.
• 2002-2003: Three children suffering from SCID syndrome developed functional immune systems after receiving hematopoietic stem cells transfused with a retroviral vector encoding a missing cytokine receptor, but developed a leukaemia-like disorder later.

Non-Viral Vectors

• Components of an ideal non-viral vector include molecules to interact with DNA, targeting moiety, endosomolytic agent, and nuclear localization sequence.

Calcium Phosphate Precipitation

• One method for non-viral delivery of DNA is Calcium Phosphate precipitation
• DNA precipitate is added to a buffer solution and dispersed into cultured cells
• Uptaken by cells via endocytosis.
• Safe, inexpensive, low toxicity; however transfection efficacy is low (less than 10% in vitro) and ineffective in vivo.

Cationic Lipids

• One of the best non-viral vectors involves cationic lipid assembly to condense DNA.
• The lipid forms multilamellar bilayer complexes that uptake DNA via endocytosis. This is an off-the-shelf method, mixing plasmids and lipids for 30 minutes, and placing it on cells.
• Downside: toxic at higher concentrations

Cationic Polymers

• Cationic polymers form polyplexes with DNA that are taken up via endocytosis
• Released from endosomes due to the "proton sponge effect."

Plasmid Fate in Vivo

• Plasmid size affects uptake and clearance.
• Leaky tumor vasculature results in reduced plasmid clearance.
• Plasmids are preferentially taken up by the lungs and liver.

Viral vs Non-viral Vectors (Comparison)

• Viral vectors are characterized by receptor binding proteins and fusogenic proteins for cell entry, and nuclear localization signals and active transport for nucleus entry
• Non-viral vectors use adsorption (+ charge) and lipid binding for cell entry, and proton-sponge effect for nucleus entry.

Anti-sense Oligonucleotides (ODNs)

• Nucleic acid strands that bind to DNA/mRNA/extracellular proteins and halt protein synthesis (inhibit transcription/translation).
• Three basic approaches are used: antisense oligonucleotides that bind to mRNA, triple helix-forming oligonucleotides that bind to duplex DNA, and oligonucleotides that bind to extracellular proteins and inhibit enzymatic activity.

Antisense Oligonucleotide Therapy

• This method inhibits protein synthesis by inhibiting transcription and translation. Using these therapies can prevent diseases from occurring.

Barriers for Efficient ON Uptake

• Lipophilic cell membranes and endosomal membranes can create barriers to the uptake of ODNs.
• Lysosomal degradation can be a problem for ODNs delivered in cells.

FDA-Approved Oligonucleotide Drugs

• Companies have developed various oligonucleotide drugs for various conditions like treating cytomegalovirus, neovascular age-related macular degeneration, familial hypercholesterolemia, Duchenne muscular dystrophy, spinal muscular atrophy, liver diseases, and Batten disease.

This quiz explores the concepts of gene delivery, gene therapy, and plasmids. It covers various vectors used in gene delivery, including viral and non-viral methods, and discusses somatic and germ line therapies. Understanding these fundamentals is essential for studying genetic engineering and therapeutic applications.