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Biomedical technology > Non-Viral vectors > Flashcards

Non-Viral vectors Flashcards

1
Q

what are the advantages to non viral vectors compared to viral vectors?

A
  • Reduced cost
  • Ease of large-scale production
  • Avoidance of the virulence commonly associated with viral delivery.
  • Quality control and probability of side effects (arising from biological contamination) much easier than viral delivery

Viral are expensive, with quality control issues and side effects

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2
Q

what are the disadvantages of non viral vectors compared to viral vectors?

A
  • Lower potency of transfection ability (+low efficiency of transfection)
  • Lower ability to traverse the various obstacles faced upon administration.
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3
Q

what can be delivered via non viral gene therapy?

A
  • Plasmids
  • microRNA
  • Short-hairpin RNA and small interfering RNAs (eg treatment of CMV infection)
  • Oligonucleotides (antisense ODN for the treatment of cytomegalovirus retinitis)
  • DNA and RNA Aptamers (Macugen, an RNA aptamer targeted against VEGF-165 and used to treat age-related macular degeneration)(The tertiary structure of the ssDNA/RNA molecule can bind to protein based on its 3d shape (aka DNA/RNA origami))
  • DNA-Protein Complexes and Ribonucleoprotein Complexes
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4
Q

give four methods of non viral gene therapy delivery

A
  • Naked DNA
  • Cationic lipids
  • Cationic polymers
  • Cationic peptides
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5
Q

what are two disadvantages to delivery by naked DNA?

A

easily destroyed and usually unable to cross cell membrane

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6
Q

give some examples of cationic lipids

A

DOGS, DOTMA, EPOPC:cholesterol
May be augmented with ferromagnetic particles (display magnetic properties so can manuver DNA to the site its needed)

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7
Q

give some examples of cationic polymers

A

PLL, PEI, chitosan

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8
Q

give some examples of cationic peptides

A

(TAT from HIV virus, GALA – synthetic peptide, HA2)

usually derived from viruses, they have approaches to escape endosomes, if we attach dna to this peptide then it will go into the cell

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9
Q

what are some extracellular barriers to non viral gene delivery?

A
  • reticularendotheial system etraptment
  • tightly packed endothelial cells prevent diffusion (loosly pack endothelial cells, like the porous leaky vessels of tumours facilitate diffusion)
  • nuclease attack
  • non-specific plasma/vessel protein interactions
  • immune system
  • skin of animals/humans
  • cellulose for plants
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10
Q

what are some intracellular barriers to non viral gene delivery?

A

binding to cytoplasmic membrane
endocytosis (escaping the endosome)
pH

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11
Q

what ways can the immune system be an obstacle to non-viral gene delivery?

A
  1. Immune responses to the delivered nucleic acids
    Cytokine induction (TNFα and IL-1β) by PEI/DNA complexes
    Lipoplex administration evoked complement activation and induction of IFN-γ, TNF-α, IL-6, and IL-12.
    Recognition of unmethylated CpG motifs by Toll-like receptor 9. Avoidable by methylating the vector DNA, using amplicons, minicircles and DNA knots.
  2. Carrier-mediated immune responses.
    Cationic lipids.
    Peptides
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12
Q

how do we target non viral particles to specific tissues

A

via attachment of specific protein

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13
Q

The Asialoglycoprotein receptor (ASGPr) target non viral vectors to what tissue?

A

Asialoglycoprotein receptor (ASGPr).
It is present on the surface of normal hepatocytes and is overexpressed in hepatocarcinoma cells.
Attachment of the ligand asialofetuin to a lipopolymeric nanoparticle targets it to the cancerous tissue

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14
Q

Transferrin targets non viral vectors to what tissues?

A

Transferrin is overexpressed in many malignancies including breast, bladder and lung.
The differential expression of the transferrin receptor and its extracellular location make it an ideal target for systemic targeting.
Systemic delivery of transferrin covalently linked to polyethylenimine has not only shown effective tumour targeting in vivo

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15
Q

what proteins can be used to target gliomas?

A

T7 peptide (HAIYPRH) was identified and shown to specifically bind to the human transferrin receptor. Utilised for targeted co-delivery of the chemotherapy drug doxorubicin (DOX) together with the human TRAIL gene (Tumour necrosis factor Related Apoptosis-Inducing Ligand) to target gliomas

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16
Q

EGF can be used to target what solid tumours?

A

EGF to target epidermal growth factor receptor (EGFR) that is upregulated in a number of solid tumours such as breast, prostate, colorectal, and ovarian. (Herceptin antibodies for HER positive breast cancer – this could be a method to EGF)

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17
Q

Fibronectin attachment protein of mycobacterium could be used to target what tissue

A

Utilised as a targeting ligand to the fibronectin molecule on epithelial cell membranes

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18
Q

what is an affibody?

A

Affibody molecules are a class of versatile non-immunoglobulin affinity proteins generated by combinatorial protein engineering.
small, robust proteins engineered to bind to a large number of target proteins or peptides with high affinity, imitating monoclonal antibodies, and are therefore a member of the family of antibody mimetics.

  • Affibody molecules have reached clinical development for inflammation disorders and in vivo tumor imaging
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19
Q

why are affibody molecules attractive for medical applications?

including intracellular application & alternative administration routes

A

small size, independence of disulfide bridges, and robust behavior

20
Q

what are the primary modes of action of affibody molecules?

A

The primary modes of action currently being explored include blocking protein interactions, inhibition of peptide aggregation, and targeted delivery of various payloads.

21
Q

What are the future possibilities for affibody molecules?

A

the possibility to develop targeted therapies, together with a companion in vivo diagnostic agent based on the same affinity protein platform, will facilitate patient stratification and monitoring of treatment efficacy in a true theranostic fashion
* Affibody molecules have reached clinical development for inflammation disorders and in vivo tumor imaging.

22
Q

what possible improvements are there to non viral delivery systems?

A
  • Capacity to efficiently interact with serum components without losing the therapeutic material.
  • Appropriate circulating time in the body and bio distribution.
  • Escape from immune system and macrophages.
  • Targeting ability of the cell.
  • Interaction with surface of cell.
  • Penetration through cell membrane barrier.
  • Intracellular trafficking capacity (release from endosomes and escape from degradation by nucleases).
  • Nuclear import capability. (if DNA is delivered to cytoplasm nothing happens, transcription happens in the nucleus)
  • Persistence in nucleus. (do we want it integrated into genome or stay as episomes)
  • Maintaining gene expression(time dependent).
  • Passage to progeny cells. (how will it pass to progeny if not integrated)
  • Ability to transcript
23
Q

how does transcriptional targeting work?

A

Many tumours have a differential expression of a particular transcription factor that can be exploited and used to restrict gene expression to a particular site.

24
Q

how can differential expression of telomerase in tumours be used in transcriptional targeting?

A

Differential expression in telomerase activity between tumour and normal tissue has enabled the use of the human telomerase reverse transcriptase and the template containing telomerase (hTERT and hTER) promoters to control gene expression.
The hTERT promoter has been used in a dual reporter system with the human alpha fetoprotein (hAFP) promoter to drive expression of MicroRNA-26a (MiR-26a), a known tumour suppressor downregulated in hepatocellular carcinoma

25
Q

how can IGF2 be used in transcriptional targeting?

A

Insulin-like Growth Factor 2 (IGF2) is involved in cellular proliferation and differentiation, but is also overexpressed in a variety of tumours such as bladder carcinoma.
The IGF2 promoter have been utilized to drive expression of the cytotoxic Diphtheria Toxin A gene

26
Q

what are the features of an ideal non viral vector system?

A
  • cationic condensing material
  • nucleic acid cargo carrying therapeutic transgene
  • tissue/tumour specific promoter
  • stealth molecule (eg PEG10K)
  • protease cleavable sequence
  • nuclear localisation signal (eg octaarginine)
  • targeting affibody (eg HER-2 antibody)
  • endosomal escape motif (eg GALA)
  • overall charge
27
Q

what is cationic condensing material?

A
28
Q

what are stealth molecules?

A

PEGylated nanoparticles are often referred as “stealth” nanoparticles, because they escape the surveillance of RES better than the control nanoparticles.

29
Q

how can non viral gene therapy be used for tissue repair and regeneration of skin?

A

Formation of a plasmid DNA/cationic polymer complex which is then loaded onto a scaffold.
Transplantation of a gene activated scaffold/matrice which fills the skin defect.
vascularization of the scaffold accompanied with repair and regeneration of the skin

30
Q

what is trigger response gene transport?

A

DNA is encased in a shell that can be destroyed by temperature, lazer, light, magnets or ultrasound
when the trigger is applied the DNA is uncaged

31
Q

give some examples of trigger responsive gene transport particles

A
  • Enz-TGR - MMP sensitive peptide onjugated PEG cationic polymer (MMP cleaves PEG off, then proton sponge effect and something)
  • L-TGR - graphene nanoparticle, trigger is NIR laser (local heat generation)
  • M-TGR - DNA polyplex with cationic polymer coated magnetic nanoparticle - trigger is applied magnetic feild (magnetic plate or bar)
  • US-TGR - nanobubble containing liposome with cationic polyplex nanoparticles (liquid to gas phase conversion (bubble breakage))
32
Q

give examples of inorganic nanoparticles commonly used for therapeutics delivery and imaging

6

A
  • quantum dot
  • silica NP
  • iron oxide NP
  • carbon nanotube
  • gold NP
  • upconversion NP
33
Q

give examples of natural nanoparticles commonly used for therapeutics delivery and imaging

A
  • exosome/ microvesicle
  • lipoprotein
  • virus-like
34
Q

give some examples of organic nanoparticles commonly used for therapeutics delivery and imaging

A

polymeric
* dendrimer
* polymeric micell
* polymeric NP

lipid-based
* liposome
* micelle
* lipid NP
* bilosome

hybrid
* polymerosome

35
Q

what forms of non viral gene delivery have been used for the genetic modification of haematopoietic stem and progenitor cells (HSPCs)?

A
  • To date, gold NPs, polymer-stabilized NPs, PLGA-NPs, lipid NPs, liposomes, virus-like particles (VLPs) and EVs have been utilized for the genetic modification of HSPCs.
36
Q

what ways can CRISPR be delivered?

A

CRISPR/Cas9 can be delivered as plasmid DNA, mRNA, or RNP complex (together with double-stranded or single-stranded DNA templates in the case of HDR), to achieve site-specific gene editing

37
Q

Delivery of CRISPR by plasmid DNA involves what?

A

Plasmid DNA needs to be delivered into the nucleus and be transcribed into mRNA, which then will be translated into Cas9 protein in the cytoplasm and be transported back into the nucleus to form a CRISPR RNP complex which can exert gene editing function.

38
Q

for mRNA delivery of CRISPR what needs to occur?

A
  • For mRNA delivery, the payload should be released in the cytosol to enable mRNA translation to protein. In contrast, CRISPR RNP need to be delivered to the nucleus
39
Q

what are the three categories for measuring invasivity? (of drug administration)

A

Category 1 - reduced invasiveness
Category 2 - minimally invasive
Category 3 - non-invasive

40
Q

Category 1 reduced invasiveness involves what methods

A

electronically driven, ballistic and combination methods
Iontophoresis, electroporation, ultrasound
gene gun, laser, magnetic feild, photomechanical waves

41
Q

Category 2 minimally invasive involves what methods?

A

micro and nano devices
microneedle assays

42
Q

Category 3 non-invasive involves what methods?

A

intelligent formulation and delivery system
nanoparticles, liposomes, emulsions, synergistic permeation enhancers
patches

43
Q

give some physical methods for gene transfer

A
  • electroporation
  • thermal assisted gene transfer
  • biolistic
  • microinjection
  • laser assisted transfection
  • ultrasound assisted gene transfer
  • hydrodynamic gene transfer
  • magnetofection
  • mechanical massage
44
Q

give four examples of laser assisted transfection techniques for gene transfer

A
  • optoinjection
  • laser influenced stress waves
  • photochemical internalisation
  • selective cell targeting via light absorbing particles
45
Q

what nanoparticles can be used for gene transfer?

A
  • gold
  • silica
  • carbon nanotubes
  • water soluble fullerenes
  • silicon nanowires
  • quantum dots
46
Q

give some chemical methods for gene transfer

A
  • calcium phosphate mediated
  • DEAE dextran mediated
  • cationic lipid mediated
  • liposomes
  • polymeres
  • nanoparticles
47
Q

give some biological methods of gene transfer

A
  • transduction using viral vectors
  • bactofection
  • virus like particles
  • erythrocyte ghosts
  • exosomes

Biomedical technology (9 decks)

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