Gene Delivery and Therapy Overview

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Questions and Answers

What is a potential risk associated with the use of retroviral vectors in gene therapy?

High specificity for target cells

Potential for insertional mutagenesis (correct)

Controlled gene expression

Efficient packing of genetic material

Which components are coded by the 'gag', 'pol', and 'env' genes of a retroviral vector?

Core proteins, reverse transcriptase, viral protein coat (correct)

Oncogenic factors, tumor suppressor genes, immune modulators

Promoter regions, enhancer sequences, packaging sequences

Viral replication enzymes, immune response genes, receptor proteins

What type of cell line is described as having essential sequences for viral replication and packaging?

Primary cell lines

Transgenic cell lines

Packaging cell lines (correct)

Immortalized cell lines

What mechanism is involved in adenoviral receptor-mediated cell entry?

Endocytosis using αv integrins and CAR Signup and view all the answers

Which of the following conditions was notably treated successfully with gene therapy in 1990?

Severe combined immunodeficiency (SCID) Signup and view all the answers

What is one common adverse effect associated with the use of adenovirus vectors for gene delivery?

Massive inflammatory response Signup and view all the answers

Which method is described for DNA delivery using calcium phosphate precipitation?

Combining DNA with calcium chloride and buffering solution Signup and view all the answers

What is a characteristic feature of cationic lipids used in gene delivery?

They assemble and entrap plasmid DNA. Signup and view all the answers

What is one challenge faced by non-viral vectors like calcium phosphate?

Ineffective transfection in vitro Signup and view all the answers

What type of disorder was induced in young children receiving retroviral vector treatment for SCID syndrome?

Lymphoproliferative disorder Signup and view all the answers

What aspect of cationic polymers influences both their transfection efficacy and toxicity?

Charge ratio and distance from backbone Signup and view all the answers

Which delivery vector is known for being the most effective at facilitating cellular entry?

Cationic lipids Signup and view all the answers

Which of the following describes a mechanism by which cells uptake DNA when using non-viral vectors?

Phagocytosis or endocytosis Signup and view all the answers

What primary role do vectors serve in gene delivery?

Vectors facilitate the entry of DNA into cells. Signup and view all the answers

Which of the following is a characteristic of viral vectors compared to non-viral vectors?

Viral vectors can cause immunogenic response. Signup and view all the answers

What is the main challenge associated with oligonucleotide delivery?

Oligonucleotides cannot enter cells due to size and stability issues. Signup and view all the answers

In what context are retroviral vectors primarily used in gene therapy?

To introduce genes into dividing cells after ex-vivo stimulation. Signup and view all the answers

What is a significant advantage of non-viral vectors over viral vectors?

Greater safety and lower risk of mutations. Signup and view all the answers

What is an essential step involved in developing a viral vector?

Integrating the viral DNA into host cell chromosomes. Signup and view all the answers

Why can't plasmids enter the nucleus of a cell on their own?

Plasmids are degraded in the cytoplasm before reaching the nucleus. Signup and view all the answers

Which of these statements regarding germ line therapy is true?

It permanently alters the genetic makeup of future generations. Signup and view all the answers

Study Notes

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.

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Description

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.