Lecture 21: Viral Genetics and Gene Therapy

Virus

A single particle (virion) that consists of a protective outer layer (capsid) encasing a DNA or RNA genome which can be single-stranded or double-stranded

• May also have a viral envelope, a second layer that helps virions attach to host cells

Viral genome contains structural genes and non-structural genes

• Structural genes — needed to assemble the virus (capsid genes and envelope genes)

• Non-structural genes — needed for replication

Structural genes

Genes needed to assemble the virus, including capsid genes and envelope genes

Non-structural genes

Genes needed for replication of the virus

Describe the general viral life cycle.

1. Virus binds to a receptor protein on the host cell membrane and is taken into the cell, usually through an endosome

2. Virus releases its genome into the host cell and the viral genome is transported to the nucleus

3. Virus uses its own viral replication genes as well as the eukaryotic host cell replication machinery to replicate and transcribe viral proteins → host cell makes copies of the virion’s capsid and envelope components

4. Virus assembles new virions with host cell-synthesized capsid and/or envelope proteins

5. The newly assembled viruses are released out of the cell or bud out of the cell

Retrovirus

A type of RNA virus that uses reverse transcription to convert its viral RNA into viral DNA for incorporation into the host DNA genome

• Contain a viral envelope coated with glycoprotein

• Contain a viral capsid protein coat

• Have a single-stranded RNA genome that contains reverse transcriptase and integrase

Describe the steps of retrovirus replication.

1. Retrovirus attaches to host cell at receptors on the host cell membrane

2. Retrovirus core containing the viral RNA enters the host cell, while viral proteins degrade

3. Viral RNA uses reverse transcriptase to make cDNA (complementary DNA) → viral RNA degrades after fulfilling its function

4. Reverse transcriptase synthesizes the second cDNA strand

5. Viral DNA enters the nucleus and is integrated into the host genome with the help of integrase, forming a provirus

6. Upon activation, proviral DNA is transcribed into viral RNA → viral RNA is exported to the cytoplasm and translated

7. Viral RNA, viral proteins, capsid coats, and viral envelopes are assembled in the cytoplasm before exiting the host cell

Provirus

When a retrovirus incorporates its viral DNA (after reverse transcription) into the host genome with the help of integrase

Integrase

An enzyme that facilitates integration of retroviral DNA into the host cell’s genome

Which of the following describes a mutation in a eukaryotic cell that would inhibit uptake/infection of the virus?

A) A nonsense mutation in reverse transcriptase

B) A silent mutation in the receptor that binds the virus

C) A frameshift mutation in a secretory protein necessary for viral budding

D) A transition mutation in the promoter consensus sequence necessary for receptor gene transcription

E) Both B and D

Answer: D) A transition mutation in the promoter consensus sequence necessary for receptor gene transcription

Which of the following describes a mutation that would inhibit a retrovirus, but not the general viral life cycle?

A) A missense mutation in reverse transcriptase that causes a neutral mutation

B) An in-frame deletion of an amino acid in the integrase enzymatic site

C) A frameshift mutation in a secretory protein necessary for viral budding

D) A transition mutation in the promoter consensus sequence necessary for receptor gene transcription

E) All of the above

Answer: B) An in-frame deletion of an amino acid in the integrase enzymatic site

Describe the two ways that retroviral infection can cause cancer.

Method 1: Proto-oncogene mutation to oncogene

• Retrovirus inserts viral RNA into the cell → retroviral RNA undergoes reverse transcription and inserts into the host chromosome next to a proto-oncogene

• When the virus reproduces, the proto-oncogene is incorporated into the virus

• After repeated rounds of viral infection and reproduction, the proto-oncogene is rearranged and mutated so that it becomes an oncogene that is inserted back into the host chromosome

Method 2: Overstimulation of proto-oncogene promoter

• Retrovirus infects a cell and the provirus inserts near a proto-oncogene

• The strong viral promoter stimulates overexpression of the proto-oncogene

Ex. Human T-cell leukemia virus type 1 (HTLV-1) causes a form of adult T-cell leukemia/lymphoma

Tumor suppressor genes

Genes that inhibit cell division in the presence of DNA damage or lack of resources

Proto-oncogenes

Genes that stimulate normal cell division

Oncogenes

Mutated, dominant-acting, stimulatory genes that cause uncontrolled cell division leading to cancer

Influenza virus

A RNA virus that has 3 main types: influenza A, influenza B, and influenza C

• Most cases of influenza A are divided into subtypes based on glycoprotein expression → hemagglutinin (H) or neuraminidase (N)

• Responsible for 1918 Spanish flu pandemic, Asian flu, Hong Kong flu, and 2009 H1N1 swine flu

How are new strains of influenza virus created?

• Antigenic shift — when a cell is infected by multiple viruses, those multiple viral genomes can recombine to create new types of viruses

  • Reassortment of RNA molecules from different strains can create new strains

Antigenic shift is the reason why we need to get new flu vaccines every year → evaluate which strain will be dominant when multiple strains are present in the same cell, and generate a new vaccine

Adenoviruses

• Medium-sized, non-enveloped virus containing a double-stranded DNA genome

• Causative agent of mild cold or flu-like illnesses

• Adeno-associated virus (AAV) vectors or cassettes are the most commonly used viral vector for gene therapy

How do scientists use adeno-associated virus (AAV) vectors for gene therapy?

• Wild-type AAV genome contains replication genes and capsid genes between inverted terminal repeats (ITRs)

• Scientists create a recombinant AAV by removing the replication genes and capsid genes and cloning in a promoter upstream of a therapeutic gene of interest

  • The replication and capsid genes are removed so that the therapeutic AAV vectors does not replicate uncontrollably within the patient

  • Once the virion is made, it cannot replicate itself

Gene therapy

An experimental technique that uses genes to treat or prevent disease — the idea is to insert a gene into a patient’s cells instead of using drugs or surgery

Describe how to clone DNA for AAV-based gene therapy.

1. Isolate genomic DNA from cells that have your DNA sequence or gene of interest.

2. Use PCR to amplify the DNA with sequence-specific primers (forward and reverse) that contain restriction enzyme sites

3. Cut the amplified DNA and AAV vector with restriction enzymes

4. Ligate the cut DNA sequence into the cut AAV vector

5. Transform bacteria with the recombinant AAV vector containing your DNA sequence

6. Purify the AAV vector DNA and verify the cloned DNA sequence is correct by Sanger sequencing

7. Transfect the AAV vector into human cells using a “helper plasmid” that contains replication and capsid genes → collect the viruses for injection into a mouse model or human patient

CRISPR-Cas9 or siRNA can also be cloned into the AAV vector, allowing for gene therapy to edit or silence dominant alleles

New viruses cannot be assembled when cells are infected with AAV-gene therapy viruses — the AAV vectors essentially stay “dormant” (the cells must be injected with helper plasmids that contain replication and capsid genes to initiate the replication of AAV vectors!)

What are some key features of the AAV vector?

AAV vector contains:

• L-ITR and R-ITR — left and right inverted terminal repeats

• CMV — a strong promoter

• MCS — a sequence containing multiple restriction enzyme recognition sites

• bGH pA — polyA adenylation site

• AmpR — antibiotic resistance gene for selection in bacteria

• Ori — origin of replication in bacteria

Transfection

The uptake of foreign DNA by plant and animal cells (eukaryotes) through artificially introducing DNA or RNA

Transformation

The uptake of foreign DNA by bacteria, yeast, and plant cells (prokaryotes) → can happen naturally or be induced artificially for genetic engineering

Compare transformation and transfection.

Transformation is for prokaryotes (and can occur naturally as well as artificially), while transfection is for eukaryotes (can only occur artificially)

Leber congenital amaurosis (LCA)

A rare type of inherited eye disorder that causes severe vision loss at birth (the most common cause of inherited blindness in childhood)

• Found in 2-3 out of 100,000 babies

• Rod cells in the retina become damaged and die

Coronaviruses (CoVs)

A highly diverse family of enveloped single-stranded RNA viruses that infect humans, mammals, avian species, livestock, and companion animals

If AAV vectors cannot replicate by themselves, what are the dangers of introducing them into patients?

Patients could have an overactive and potentially deadly immune response to the viral load

Describe how Kary Mullis’ 1984 invention of Polymerase Chain Reaction (PCR) allowed researchers to synthesize mRNA.

PCR is a method to amplify DNA

• Heat DNA to 90-100°C to separate strands

• Cool DNA to 30-65°C to allow short single-stranded RNA primers to anneal

• Heat DNA to 60-70°C to allow DNA polymerase to synthesize new DNA

• Repeat for as many cycles as needed — each time the cycle is repeated, the amount of target DNA doubles (increases exponentially)

PCR can be modified to synthesize mRNA — use RNA polymerase instead of DNA polymerase!

Describe Dr. Drew Weissman’s and Dr. Katalin Kariko’s discovery of how mRNA can be expressed in mammalian cells without triggering the innate immune system response.

Kariko was trying to inject mRNA into mammalian cells, but she couldn’t understand why her experiments weren’t working

• Weissman suggested that the reason why mRNA was being destroyed by the cells was because mRNA activated the cells’ innate immune response

• Toll-like receptors detect microbial products in mammals and activate the innate immune response → toll-like receptor 7 (TLR7) recognized ssRNA

• Had to find a way to modify ssRNA so that it wouldn’t activate TLR7 → which nucleotide in ssRNA activates the innate immune response?

• Uracil in foreign ssRNA activated the response — uracil in innate ssRNA doesn’t activate the response because it is modified as pseudouridine

Would gene therapy work better for a recessive disease or a dominant disease?

Gene therapy only works for recessive diseases, because if you are supplying a wild-type form of the gene, then the recessive allele can be suppressed by the therapeutic allele, but a dominant allele will still have a dominant effect and cannot be suppressed by the therapeutic allele

What is one of the major challenges with viral gene therapy?

• Accurate and targeted delivery of the AAV vectors to a specific organ or tissue in the cell

• A large gene of interest that is too big for the AAV vector to package