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Biomedical technology > Viral vectors > Flashcards

Viral vectors Flashcards

1
Q

what are the ideal requirements for the delviery of genes?

4

A
  1. Safe
  2. Efficient uptake of DNA into host cell
  3. Long term expression
  4. High levels of expression
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2
Q

why are viruses the preferred vehible for gene transfer?

A

they are highly efficient at introducing DNA into cells

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3
Q

give 5 examples of viruses used as vectors

A

retroviruses, adenoviruses, herpesviruses, vaccina virus, adeno associated virus

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4
Q

give four features of viruses

unique feature, genome, structure

A
  • Intracellular parasites that cannot replicate on their own
  • All viruses contain a nucleic acid genome
  • Can be single stranded or doubled stranded DNA or RNA
  • The genetic material is surrounded by a protein coat known as a capsid
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5
Q

what is the most studied viral vector system?

A

retroviral vectors

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6
Q

what is the retroviral genome like?

A

ssRNA genome 7-9kb

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7
Q

what are the five integral components of the retroviral genome?

A
  • LTR – long terminal repeat – contains all the control regions
  • Ψ – packaging signal
  • GAG – encodes for capsid
  • POL – encode reverse transcriptase
  • ENV - encodes the envelope protein
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8
Q

what does GAG encode?

A

the capsid

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9
Q

what does POL encode?

A

reverse transcriptase

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10
Q

what does ENV encode?

A

envelope protein

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11
Q

what is reverse transcriptase?

A

a DNA polymerase enzyme that transcribes single-stranded RNA into DNA.

reverts the ssRNA to dsDNA so it can integrate into the host genome

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12
Q

what is required for the proteins to be packaged?

A

packaging signal

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13
Q

descibe the basic infection and reproduction of a virus with ssRNA genome

A

entry into cell and loss of envelope
reverse transcriptase reverts viral ssRNA into dsDNA whic then integrates in host chromosome
this integrated DNA is then transcribed and translated along with the rest of the genome
the new capsid, envelope and reverse transcriptase proteins are then packaged and assembled into new virus particles

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14
Q

describe the packaging process of a retrovirus

A

the capside containing the viral chromosome (nucleocapsid) approaches the lipid bilayer wher transmembrane viral envelope proteins site
the nucleocapside induces assembly of envelope proteins
th particl then begins to bud off the membrane and we get a new progeny virus

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15
Q

most retroviral vectors are based on MoMLV (Moloney Murine Leukaemia virus) - what does this genome look like?

A

the coding region of CAG POL and ENV is removed
all that remains are the LTR and packaging signal along with the gene insert
(if retained the resulting RNA would be too long to be packaged (7-9kb)

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16
Q

how do we package the viral vectors?

A

use a packaging cell line
( the packaging plasmid contain the viral packaging components while the other plasmid carries your gene of interest )

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17
Q

what does the genome of the packaging cell lines look like?

A

LTRs, GAG, POL, ENV
but NO packaging signal

no packaging signal so RNA jujst accumulates

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18
Q

when was the first retrovirla vector used?

A

First used in 1990 to treat a young girl with ADA deficiency

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19
Q

what is ADA deficiency?

A
  • inherited disorder caused by defective adenosine deaminase (ADA) gene causing damage to the immune system
  • ADA encodes for a gene in the purine salvage pathway
    • T lymphocytes most severely affected
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20
Q

describe how retroviral vectors were used to treat Ashanti DeSilva

ADA deficiency

results?

A

collected T lymphocytes from ADA- patient, and recombinant ADA retrovirus vector created
expansion of T cells in culture and infections with ADA recombinant
Integration to give some ADA+ cells
selection of ADA+ cells
transfusion into ADA deficient patient
expression of induced ADA genes can overcome ADA deficiency

results: trial was not pure, patients also treated with purified ADA protein, however ADA from the retroviral vector was produced in her blood cells, good expression of ADA as a result reducgin the amount od ADA protein they gave her

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21
Q

what is X SCID?

buble boy

A

X linked severe combined immunodeficiency syndrome)
Caused by a mutation in IL2RG. - lack of correct cytokine receptor gene
Results in a near-complete absence of T and natural killer (NK) lymphocytes and nonfunctional B lymphocytes.
almost universally fatal in the first two years of life
blocks the differentiation pathway (lymphocyte production, survival and function)

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22
Q

what was the serious side effects of SCID-X1 gene therapy?
give statistics

A

some children treated with retroviral therapy developed acute lymphoblastic leukeamia – the gene had integrated next to a protooncogene

in france out of 9 teated, 4 developed T ALL and 1 died
in UK 10 were treated and 1 developed T ALL

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23
Q

what is insertational mutagenesis?

A

the phenomenon by which an exogenous DNA sequence integrates within the genome of a host organism. This event can result in the deregulation of genes in the neighborhood of the insertion site and can potentially cause a perturbation of cellular phenotype.
can cause high level of contituative expression of protooncogene

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24
Q

what are the advantages and disadvantages of retroviral therapy?

A

Advantages
1. High efficiency of gene transfer
2. High levels of expression
Disadvantages
1. max insert size 7-7.5kb
2. only infects dividing cells
3. Toxic?

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25
Q

give two important features of adenoviral genome

A
  • DNA virus
  • 36kb of ds DNA
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26
Q

breifly describe the life cycle of adenoviruses

A
  1. Early genes are expressed E1a and E1b
  2. Expression of E2-E4 followed by viral DNA replication
  3. Expression of late genes
  4. Packaging of new virus particles and cell lysis
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27
Q

what is the structure of the first adenoviral vectors?

A

E1 removed to allow insertion of transgene and to stop viral replication
Remove coding region of E1a E1b and replace with the protein we want to express

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28
Q

describe the first adenovirus trial

otc

A

First adenovirus trial – Jesse Gelsinger
In 1999, an 18-year-old patient with ornithine transcarbamylase deficiency - it impairs the elimination of ammonia.
Jesse was heterozygous so was treated with a low-protein diet and drugs.
On Sept. 13, 1999, Gelsinger was treated with the corrective OTC gene via an adenovirus vector
Gelsinger experienced a severe immune reaction to the vector and died 4 days after receiving the injection.
- Low level of transcription from viral genes
- Led to systemic activation of the innate immune response

Most infants with OTC experience severe brain damage & usually die before the age of 5 years

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29
Q

what were the findings from the investigation of the frist adenovirus trial?

A

Screening of patients – had liver problem and should have been excluded
Informed consent: past research subjects as well as experimental animals had become sick and died from the vector.
A conflict of interest: lead scientist had a financial interest in the development of the adenovirus vector being used in the OTC gene therapy trial.

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30
Q

describe the second generation adenoviral vectors

A
  • have deletions of E1, E2, and E4 genes, because viral proteins encoded by these genes were shown to induce most of the host immune response.
  • This does fully disable the virus, no expression of viral DNA – much better tolerated in the human body

Currently 2 stage III clinical trials using expression of p53 in advanced ovarian cancer
These vectors have decreased toxicity and prolonged gene expression

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31
Q

what are the advantages and disadvantages of adenoviral vectors?

A

advantages
- do not insert the gene into the host chromosome
- taken up by epithelium derived tissues - good expression in the liver, heart, lung, brain and goof for solid tumours

disadvantages
- limited to only temporary protein expression
- not so good for haematological tumours

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32
Q

approximately what percentage of the western world carry the herpes simplex virus

A

70%

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33
Q

what are the three types of gene transfer method?

A
  • physical methods
  • chemical methods
  • biological methods
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34
Q

what is the manifestation of the herpes simplex virus?

A

a cold sore

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35
Q

why is herpes simplex virus good for gene therapy?

A
  • its well studied
  • large dsDNA genome 150kb
  • carries 80 genes but not all are required for viral propagation - some can be safely deleted
  • TBD kinase stops proliferation of virus (most commonly used suicide agent in gene therapy for cancer)
  • the viral particle itself have a capsid and envelope
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36
Q

describe the propagation of herpes simplex virus

A
  1. after attachment, the host cell membrane fuses with the viral envelope, thereby permitting enty of the nucleocapsid to the cytoplasm
  2. the viral capsid is uncoated and the DNA od the viral genome enters the cells nucleus
  3. new viral DNA is synthesised in the nucls
  4. transcription produces mRNAs that are translated on cytoplasmic ribosomes into capsid and spike proteins
  5. capsid proteins enter the nucleus and combine with viral genomes and to form new nucleocapsids
  6. the viruses bud through the nuclear membrane but do not acquire their final envelop and spikes until reaching a golgi compartment in the cytoplasm
  7. exocytosis releases new virons
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37
Q

which types of cell does the wt herpes simplex virus replicate in ?

A

The wild-type virus replicates in epithelial cells of the skin or mucosa at the site of contact,

and progeny particles are taken up by the terminals of sensory neurons innervating the infected epithelium.

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38
Q

what types of cell takes up the progeny of the herpes simplex virus?

A

progeny particles are taken up by the terminals of sensory neurons innervating the infected epithelium.

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39
Q

what is an episome?

A
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40
Q

hows does the herpes simplex virus persist for life?

A

once the manifestation (cold sore) is gone, the viral DNA stays in the sensory neurons and stays dormant
only 2 genes are working, L1 and L2 - these encode non coding RNA

the promoters of these two genes are useful to engineer genetically modifying virus because they enable us to create consistent expression for life

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41
Q

give four unique features that make HSVs advantagous for gene therapy

A
  • Very high transgenic capacity of the virus particle allowing to carry long sequences of foreign DNA
  • Genetic complexity of the virus genome, allowing to generate many different types of attenuated vectors possessing oncolytic activity
  • Ability of HSV vectors to invade and establish lifelong non-toxic latent infections in neurons from sensory ganglia from where transgenes can be strongly and long-term expressed.
  • Latency active promoters LAP1 and LAP2 may be used to drive long-term transgenic expression in neurons of the peripheral nervous system

capacity of virus to take payload is quite high
able to generate many variants
can be reengineer to target specific tissues (must be validated)

42
Q

what are the disadvantages of using HSVs for gene therapy?

5

A
  • Fairly hard to produce in vitro mass production
  • Must make very pure
  • Requires preservation of viral genes
  • Highly immunogenic – majority not population already has antibodies against virus because majority already has it, this is dangerous
  • Natural tropism to neurons
43
Q

what modifications can be made of the herpes simplex virus to make it a tool for gene therapy?

3

A
  • Replication modifications. Necessary to avoid uncontrolled infection. (replication deficient or not at all)
  • Re-targeting.
  • Insertion of therapeutic transgene or a number of transgenes.
44
Q

what replication modifications can be made to HSV?

A
  • amplicon vectors
  • replication-defective viruses
  • genetically engineered replication-competent viruses with restricted host range
45
Q

what are amplicon vectors?

A

Amplicons are bacterial plasmids that contain transgene cassettes and two non-coding viral sequences, an origin of DNA replication (ori) and a DNA cleavage/packaging signal (pac) and they require a helper system to be produced.

it needs a viral vector to carry the rest, eg the ones needed for replication

46
Q

what are the advantages of amplicon plasmids?

A
  1. A large transgene capacity (150 kb)
  2. Repetitive character of the genome carried by the amplicon particle ensures the introduction of multiple copies of the transgene per infected cell
  3. Ability to infect a wide variety of cell types, including dendritic cells
  4. Ease of vector construction
  5. limited toxicity due to lack of viral coding sequences

works great in the lab however difficult to mass produce - variation, impurities, danger of creating the live virus

47
Q

what are replication defective vectors?

A
  • The replication-defective viruses are viral vectors where “essential” genes for in vitro viral replication are either mutated or deleted.
  • These mutants cannot grow except in transformed cell lines, where they are complemented in trans.
48
Q

what are attenuated vectors?

A
  • Deletion of some non essential viral genes results in viruses that retain the ability to replicate in vitro, but are compromised in vivo - Replication inefficient (To limit the infections rate)

Always try and preserve tmd kinase gene so you have a kill switch

49
Q

what ways have attenuated HSV vectors been tested?

A

as live viral vaccines, as oncolytic viruses and as gene therapy vectors to deliver transgenes to the nervous system

50
Q

give 3 ways HSV-1 can be used as a vector therapeutically

A
  • vaccination
  • gene therapy for the nervous system
  • gene therapy of cancer
51
Q

why is herpes simplex virus 1 good for use in vaccinations?

A
  • Elicit strong and durable immune responses by various routes of inoculation
  • Viral DNA persists inside the host’s cell nucleus as an episomal element, thus eliminating the safety concerns deriving from the random integration of the viral genome into the host’s DNA
  • Carry the tk gene, that, in case of undesired effects, can be used, in combination with specific antiviral drugs, to kill the virus-harboring cells.

all types of HSV vectors have been used for developing vaccines

52
Q

what types of HSV vectors are there?

A
  • amplicon vectors (HSV is helper for amplicon plasmid)
  • replication defective vectors
  • attenuated vectors
53
Q

why is HSV good for gene therapy of the nervous system?

A

HSV-1 contains genes that control neuro-invasiveness and neurovirulence. The virus can move both in the retrograde and anterograde directions and disseminates trans-synaptically from neuron to neuron

54
Q

how can gene therapy using amplicon vectors help with neuroprotection against brain injuries?

A

transduction of cells with amplicons expressing
- neurotrophins
- anti-apoptotic proteins
- antioxidants
- other proteins

55
Q

how can gene therapy using amplicon vectors help with neurodegenerative disorders like parkinsons disease?

A

transduction of cells with amplicons expressing
- dopamine biosynthetic enzymes or/and vesticular monoamine transporters
- neurotrophic factors

56
Q

how can gene therapy using amplicon vectors help with inherited neural genetic disorder like ataxia-telangiectasia?

A

transduction of cells with amplicons expressing the cDNA of ATM gene

57
Q

how can gene therapy using amplicon vectors help with inherited neural disorders like Friedreich’s ataxia?

A

transduction of cells with amplicons expressing the FRDA human genomic locus

58
Q

give two ways we can use gene therapy for alzheimers disease?

A
  • silencing of NR1-NMDAR subunit expression through delivery of NR1 antisense sequences
  • vaccination against Abeta peptides to prevent or remove peptide deposition
59
Q

what potential gene therapy targets could be used to target glioblastomas?

A
  • inhibitors of metalloproteinases to inhibit invasive activity
  • silencing of the EGFR gene
  • expression of FasL, FADD, TRAIL to induce apoptosis in cancer cells
60
Q

how can oncolytic viruses kill cancer?

A
  1. Direct oncolysis or apoptosis of cancer cells.
  2. Apoptotic death of uninfected cells (natural cellular defence mechanism to limit viral spread).
  3. Induction of an immune response
61
Q

what is the only oncolytic viral therapy to be approved?

(2015)

A

Talimogene laherparepvec (T-Vec) for melanoma treatment. FDA approval 2015

62
Q

describe the process of infections and killing by oncolytic viruses

A
  1. administration and infection of tumur cells
  2. viral replication
  3. tumour cell lysis
  4. replicated viruses infect nearby tumour cells (cycle)
  5. release of tumour derived antigens (TDAs)
  6. uptake, processing and presentation of TDAs by antigen presenting cells (APCs)
  7. activation and priming of T cells by APCs
  8. effector cells localise to and infiltrate tumour
  9. effector cells recognise and kill tumour cells with subsequent release of TDA
  10. cycle continues
63
Q

what other cancers has T-Vec been evaluated for?

A
  • Other studies have been conducted to evaluate T-VEC in pancreatic cancer, hepatocellular carcinoma and non-melanoma skin cancers, breast cancer, head and neck squamous cell carcinoma
64
Q

what was the outcome of the T-Vec trials for squamous cell carcinoma of the head and neck?

A

A small trial of 17 patients with stage III or IV squamous cell carcinoma of the head and neck was conducted with T-VEC in combination with cis-platinum chemotherapy and radiation therapy followed by surgery

Fourteen (82.3%) of patients demonstrated objective responses by imaging or clinical exam with 93% showing pathologic complete response at the time of surgery.
At a median follow-up of 29 months, disease-specific survival was seen in 82.4% of patient

(Harrington et al., 2010).

65
Q

what was the outcome of the T-Vec trial for breast cancer?

A

T-VEC was tested in breast cancer patients with inoperable locoregional recurrence

Nine patients were enrolled and six patients had locoregional disease only and three had additional metastatic lesions. While no significant adverse events were reported, no patients had an objective response.

(Kai et al., 2021).

66
Q

what is T-Vec?

A

a genetically modified live oncolytic herpes simplex virus therapy that is intended to treat melanoma lesions that cannot be removed completely by surgery.

The therapy is injected directly into melanoma lesions, increasing selectivity for cancer cells and secreting cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) to promote an immune response. The virus invades both cancerous and healthy cells, but is unable to replicate in healthy cells, leaving them unharmed

67
Q

what are the two main effects oncolytic viruses have on tumours?

A

Local effect: tumour cell lysis
Systemic effect: tumour specific immune response

68
Q

What immunomodulating gene is used in HSV-1 based therapies?

A

IL-12

69
Q

what antiangiogenic genes are used in HSV-1 based therapies?

A

angiostatin
or
angiostatin + IL-12

70
Q

what proapoptotic genes are used in hSV-1 based therapies?

A

secretable TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)

71
Q

give three therapeutic transgenes that could be used in HSV-1 based therapies for cancer

A
  • Immunomodulating genes: IL-12
  • Anti-angiogenic genes: Angiostatin or Angiostatin + IL-12
  • Pro-apoptotic genes: secretable TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)
72
Q

give 4 reasons why adeno associated vectors are used

A

Not a single disease has been associated with adeno associated virus (infects humans and some other primates but causes no known disease)
13 serotypes so can infect virtually any type of tissue
is a DNA virus so is generally maintianed extrachromosomally (if itdoes integrate into human genome it does so at a specific place on chromsome 19)

73
Q

do adeno associated viruses integrate into the human genome?

A

AAV is a virus that is generally maintained in an extrachromosomal form.
Wild-type AAV is known to integrate into the AAVS1 site of the human genome, however, this is mediated by the Rep protein and AAV vectors used in gene therapy lack the Rep protein

74
Q

what are the advantages and disadvantages of using adeno associated viruses for gene therapy?

A

Advantages
1. Well tolerated
2. Easy to manipulate
3. Non-pathogenic
4. Can infect a wide variety of normal and cancer cells (dividing and non-dividing).
5. Ability to mediate stable and long-term gene expression.

Disadvantages:
1. Limited capacity to carry therapeutic genes.
2. Most of the people were already exposed to AAVs and developed neutralizing antibodies.

75
Q

describe AAV-mediated gene therapy

A
  1. virus binds to receptor
  2. endosome encapsulation
  3. endosomal escape and translocation into nucleus
  4. uncoating - viral ssDNA
  5. transcription of the episomal DNA into mRNA
  6. move out of nucleus for transgene translation
  7. trangenic protein produced
76
Q

what are the immunological barriers to rAAV delivery?

A
  1. The recombinant adeno-associated virus (rAAV) may encounter neutralizing antibodies (NAbs) that are widely found in the human population.
  2. The rAAV capsid and genome may trigger innate immunity via activation of Toll-like receptor 2 (TLR2) and TLR9, respectively.
  3. The capsid undergoes proteasomal degradation, and the resulting peptides are presented by major histocompatibility complex (MHC) class I molecules to CD8+ T cells. The CD8+ T cell can exert destructive cytotoxic effects to eliminate rAAV-transduced cells, resulting in the loss of transgene expression.
  4. The transgene product can elicit a humoral immune response to generate transgene product-specific antibodies that can compromise therapeutic efficacy.
77
Q

give a couple of examples how AAV vectors can be used against cancer

A
  • anti-angiogenesis
    AAV1 for ovarian cancer with VEGFR-1, AAV2 for bladder and pancreatic cancer with endostatin, AAV5 for lung cancer with vasostatin
  • cytotoxic or suicide genes
78
Q

give four gene therapy approaches for cardiac diseases

A

plasmids
adeno associated virus
lentivirus
adenovirus

79
Q

compair the gene therapy approaches for efficacy of treatment of cardiac diseases

A

plasmid - low cardiac transfections, expression up to 2 months, low transfection efficacy

AAV - dsDNA4.8kb, cardiotropic AAVserotypes, long term cardiac expression, risk of neutralising antibodies and t cell responses

Lentivirus - RNA 10kb, low cardiac transfection, long term cardiac expression, risk of insertation mutagenesis

Ad - dsDNA 36kb, high cardiac transduction, expression up to 2 weeks, high antibody and inflammatory response

80
Q

what is the role of cholesterol in cardiac disease?

A

With high cholesterol, you can develop fatty deposits in your blood vessels. Eventually, these deposits grow, making it difficult for enough blood to flow through your arteries. Sometimes, those deposits can break suddenly and form a clot that causes a heart attack or stroke
** A 10% decrease in total blood cholesterol levels can reduce the incidence of heart disease by as much as 30%. **

81
Q

give three examples of potential targets for genetic modifications in the liver that could help in the treatment of cardiovascular diseases

A
  • Proprotein convertase subtilisin/kexin type 9 (PCSK9)
  • Angeiopoietin-like 3 (ANGPTL3)
  • Apolipoprotein C-III (ApoC3)
82
Q

what is Proprotein convertase subtilisin/kexin type 9 (PCSK9)?

A

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted enzyme that binds to the low-density lipoprotein receptor (LDLR) and promotes its degradation.

Gain-of-function mutations in the PCSK9 gene result in familial hypercholesterolemia (FH), a disease with dramatically elevated low-density lipoprotein (LDL) cholesterol levels, due to an inability of the LDLR to recycle back to the cell surface.

83
Q

what is angiopoietin-like 3?

A

Angiopoietin-like 3 (ANGPTL3) is a protein that inhibits lipoprotein lipase (LPL), the major enzyme responsible for clearance of triglycerides from the circulation.

Loss-of-function mutations in ANGPTL3 in humans are associated with decreased cholesterol, LDL, triglycerides, and reduced risk of CVD , providing strong genetic rationale for inhibition or disruption.

84
Q

what is Apolipoprotein C-III?

A

Apolipoprotein C-III (ApoC3) inhibits LPL and is a negative regulator of triglyceride metabolism.

Loss-of-function mutations in APOC3 in humans result in lower triglycerides and reduced CVD risk.

85
Q

give some examples of potential transgenes and their effects on tumours?

AAVs

A

activation of tumour suppressors: PTEN, TP53

silencing of oncogenes: miRNAs targetting MYCN, MYC, WNTs

cell death induction: TRAIL, FASL, miR26a, HSV1-TK

cell cycle arrest: CDKs, Cylcins, miR-122, MIS

targeted immune response: IL15, IL12, IL27, DNase1, CAR-T

anti-angiogenesis: sVEGFR1, VEGF-TRAP, sVEGFR3, Bevacizymab

86
Q

give examples of four tumour tropic AAVs

A

AAV3 for HCC
AAV5 for lung cancer
AAV8 for melanoma
rh.10 for ovarian

86
Q

give examples of four tumour tropic AAVs

A

AAV3 for HCC
AAV5 for lung cancer
AAV8 for melanoma
rh.10 for ovarian

87
Q

give examples of 3 universal AAVs

A

AAV1
AAV2
AAV9

88
Q

give an example of an organ tropic AAV

A

AAV6 for skeletal muscle

89
Q

give 3 examples of ubiquitous promoters

A

CBA
U6
CMV

CBA, chicken beta actin.
U6, type III RNA polymerase III.
CMV, cytomegalovirus.

90
Q

give two examples of tissue specific promoters

A

hAAT
NSE

hAAT, human alpha 1 antitrypsin.
NSE, neuron-specific enolase

91
Q

give four examples of cancer specific promoters

A

hTERT
CXCR4
TNC
AFP

hTERT, human telomerase reverse transcriptase.
CXCR4, C-X-C motif chemokine receptor
TnC, tenascin-C
AFP, alpha fetoprotein.

92
Q

what are some of the challenges in AAV manufacturing and production?

A

difficulties in upstream processing for AAV production at the clinical scale.
* Adherent cells are not scalable due to their growth on surfaces.
* suspension cells grow in 3D and so represent a more scalable approach, but have poor transfection quality (developing efficacious suspension cell lines is not a trivial task.)

difficulties in downstream processing for AAV production at the clinical scale.
* Mechanical cell lysis can cause losses via AAV aggregation while chemical cell lysis introduces toxic contaminants.
Chromatography-based purification methods are much more scalable than ultracentrifugation, but these approaches come with certain drawbacks
* Affinity chromatography cannot separate empty capsids from full capsids.
* Ion-exchange chromatography can remove some of the empty capsids, but also loses many of the full capsids due to poor specificity and the need for extreme pH conditions.

93
Q

describe the workflow of AAV production at the small laboratory scale

A
  1. triple transfection with pHelper, pRap-Cap, pTransfer
  2. cellular production
  3. lyse cells
  4. ultracentrifugation on CsCl or iodixanol gradient
  5. extract AAV band
  6. dialyses to remove CsCl or iodixanol
  7. purified AAVs
94
Q

give two alternative established methods for AAV production

A

transduction of insect cells by engineered bacula viruses (carrying AAV components)(using three vectors)

transduction of mammalian cell with engineered HSVs (carrying AAV components)(using two vectors)

95
Q

give three emerging synthetic biology approaches for AAV production

A

Yeast platforms for AAV production have been explored, though they suffer from very low yields and poor AAV potencies. Synthetic biology engineering of yeast has the potential to ameliorate these issues and pave the way for establishing yeast as a low-cost production system for AAV manufacturing.

TESSA utilizes helper adenoviruses (TESSA-AAV and TESSA-Rep-Cap) which contain designed genetic circuits. These engineered adenoviruses repress production of their own capsid proteins except in the presence of doxycycline. TESSA achieved 30-fold higher yield than triple transfection without generating infectious adenovirus or replication-competent AAV contamination.

In vitro production of dbDNA may act as a more easily scalable alternative to AAV plasmids. The dbDNA synthesis process leverages Phi29 DNA polymerase for rolling circle replication and protelomerase for covalently closing the ends of target sequences flanking AAV DNA. Since the final dbDNA can denature into a circle of linear ssDNA with terminal protelomerase binding sites, it acts as the substrate for further amplification, enabling easy production of large amounts of the product.

96
Q

what is rolling circle replication?

A

a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA,

97
Q

describe the process of rolling circle replication

A

Rolling circle DNA replication is initiated by an initiator protein encoded by the plasmid or bacteriophage DNA, which nicks one strand of the double-stranded, circular DNA molecule at a site called the double-strand origin, or DSO.
The initiator protein remains bound to the 5’ phosphate end of the nicked strand, and the free 3’ hydroxyl end is released to serve as a primer for DNA synthesis by DNA polymerase III.
Using the unnicked strand as a template, replication proceeds around the circular DNA molecule, displacing the nicked strand as single-stranded DNA.
Displacement of the nicked strand is carried out by a host-encoded helicase called PcrA (the abbreviation standing for plasmid copy reduced) in the presence of the plasmid replication initiation protein.

98
Q

give an example of an engineered hybrid virus

A

PVSRIPO, an engineered hybrid of poliovirus and rhinovirus that cannot attack neurons but retains cytotoxicity in neoplastic cells, including glioma

99
Q

how can vaccina virus be used in gene therapy?

A

JX-594 expressing GM-CSF in hepatocellular carcinoma.
75% survival of treated patients vs 18% of untreated in 12 month.

a replication-competent Wyeth strain vaccinia virushat was genetically modified to inactive the endogenous thymidine kinase gene and to express human GM-CSF and LacZ genes.

100
Q

what is the seneca valley virus (SVV-001)?

A

Naturally occurring ssRNA replication-competent picornavirus with potent and selective tropism for neuroendocrine cancer cell types, including small cell lung cancer.
Promising vector for glioblastoma and medulloblastoma treatment

Biomedical technology (9 decks)

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