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Adeno associated virus > AAV general > Flashcards

AAV general Flashcards

adeno associated virus - facts

1
Q

AAV vector genome

A 
ssDNA genome
4.7kb
consists of
ITR-Rep-Cap/AAP-ITR
three promotors (p5, p19, p40) 
nine gene product; alternative translation start sites, differential splicing
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2
Q

large Rep proteins

A

Rep 78 and 68
under p5 promotor
mediate integration of wtAAV genome
bind to Rep-binding elements within ITR’s to mediate integration
they unwind the DNA and introduce a single-stranded nick at the adjacent terminal resolution sites (trs)

problem: known to induce DNA damage, cell cycle arrest and apoptosis

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3
Q

AAV structure

A

non-enveloped

icosahedral

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4
Q

AAV abbreviation
family
infectivity

A

adeno associated virus
parvovirus family
dependent on co-infection

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5
Q

AAV discovery

A

found as a contamination of adeno virus preparations

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6
Q

Rep

A

encodes for family of non-structural proteins
needed for viral genome replication and packaging
named by size
Rep 78,68,52,40

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7
Q

small Rep proteins

A

Rep 52 and 40

under p19 promotor

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8
Q

Cap

A 
encodes for capsid proteins
VP1,VP2,VP3
all share common VP3 region
under control of p40 promotor
ratio 1:1:10
share the same stop codon
defines the tropism and protect the DNA genome
forms the icosahedral protein shell, consists of 60 subunits
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9
Q

VP1

A

87kDa

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10
Q

VP2

A

72kDa
unnatural start codon: ACG
decreased translation

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11
Q

VP3

A

62kDa
strong start codon: AUG
increased translation in comparison to VP2

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12
Q

AAP

A

assembly activating protein
alternate reading frame in Cap
nuclear protein
thought to provide scaffolding function for assembly
essential for nuclear localization of VP proteins
non-essential in AAV4, 5 and 11

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13
Q

ITR

A 
inverted terminal repeats
flank recombinant AAV genome
origin of replication and packaging signal
145 nucleotides
125 first nucleotides are palindromes
T-shaped
contain Reb-binding elements
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14
Q

recombinant AAV

A

replication deficient
lack viral DNA, only ITR’s left
“protein-based nanoparticle”
transgene remains as episomes in absence of Rep in the nucleus of a transfected cell
does not integrate, lost quickly through cell division
long lasting expression in non-dividing cells
random integrations happen only very rarely

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15
Q

loss of transgene expression

A

rAAV does not integrate
is out-diluted in dividing cell
time dependent on turnover rate of specific cell type

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16
Q

actively dividing cell types

A

stem cells

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17
Q

limitations of rAAV vector design

A

packaging size

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18
Q

limitations of rAAV vector design

A

packaging size

broad tropism, loss of strong transgene expression through outdilution into non-target tissue

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19
Q

strategies to increase coding capacity

A

overlapping vectors

dual vectors

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20
Q

strategies to increase coding capacity

A

overlapping vectors
dual vectors
co-injection of two vectors

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21
Q

self complementary transgene

A

scAAV
packaged ssAAV genome complements itself to form dsDNA
accomplished through a mutation in one ITR
faster transgene expression
only half of the coding capacity available (around 3.3kb left)

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22
Q

AAV2

A

one of the first serotypes to be characterised
best “lab model”
most rAAV’s use ITR’s of AAV2

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23
Q

AAV2

A

one of the first serotypes to be characterised
best “lab model”
broad tropism, accumulation in liver tissue
most rAAV’s use ITR’s of AAV2
HSPG as primary cell surface receptor
fibroblast/hepatocyte growth factor receptor and the integrins aVb5 and a5b1 as secondary cell surface receptors
can induce a latent infection through preferential integration into human chromosom 19 (19q13.24) = AAVS1 locus

24
Q

CMV promotor

A

cytomegalovirus promotor/enhancer
strong constitutively active promotor
length around 800bp
known to be silenced in CNS (central nervous system)

25
Q

commonly used strong promotors

A

CMV
EF1a
SV40
CAG (strongest expression, but also largest construct 1.7kb)

strong constitutively active expression in most cell types
can be subjected to silencing in certain cell types

26
Q

How to restrict transgene expression to a certain target tissue?

A

usuage of tissue specific promotors
usuage of capsids who show a certain tropism prefference
may be enhanced by capsid engineering

27
Q

How to restrict transgene expression to a certain target tissue?

A

usuage of tissue specific promotors
usuage of capsids who show a certain tropism prefference
may be enhanced by capsid engineering
codon engineering helps to maximize expression in desired tissue

28
Q

promotor specific for cardiac muscle

A

alpha-myosin heavy chain promotor

29
Q

promotor specific for cardiac muscle

A

alpha-myosin heavy chain promotor

30
Q

promotor specific for neurons

A

enolase promotor

31
Q

promotor specific for liver

A

alpha1-antitrypsin promotor

32
Q

AAV9

A

uses galactose as primary cell surface receptor
unique ability to cross blood-brain-barrier and transduce cells of CNS, including primary neurons
effictive to transduce skeletal and cardiac muscle

tropism: liver + skeletal muscle + lung
receptor: galactose + laminin receptor

33
Q

primary cell surface receptors

A

pCSR
usuage differs greatly between AAV serotypes
initial interaction between AAV capsid and sugars on cell surface
typically sialic acid, galactose and heparin sulfate

34
Q

secondary cell surface receptor

A

sCSR

usuage differs greatly between AAV serotypes

35
Q

AAV6

A

performs best in stem cells (CD34+ cells)

tissue tropism: skeletal muscle + lung
receptor: siliac acid + heparan sulfate proteoglycan (pCSR) + epidermal growth factor receptor as sCSR

36
Q

AAV5

A

tissue tropism: neuronal + retinal pigmented epithelia + photoreceptors
receptors: siliac acid (pCSR) + platelet-derived growth factor receptor as sCSR

37
Q

AAVR

A

type I membrane protein

recently identified as a critical receptor for AAV cell binding and internalization

38
Q

AAV8

A

efficiently transduces liver of rodents and non-human primates
used in clinical trials hemoglobinopathies

tropism: liver + skeletal muscle + heart + pankreas
receptor: laminin receptor

39
Q

AAV1

A

efficient in transducing skeletall and cardiac muscle
used in clinical trial of heart failure
approved for treatment of lipoprotein lipase deficiency

tissue tropism: neuronal + skeletal muscle
receptors: sialic acid

40
Q

capsid engineering approaches

A 
capsid shuffling
directed evolution
random peptide library insertions
in-vivo directed evolution
inclusion of larger binding proteins on the capsid (DARPins)
41
Q

DARPin

A

designed ankyrin repeat protein
used in rAAV capsid design to convey certain tropism
includes e.g. portions of protein A or cytokines
successfully used to target CD4+ cells and tumor cells

42
Q

infection pathway of wtAAV

A 
binding to pCSR and sCSR
clathrin mediated internalization into endosomes
trafficing through endosomes
endosomal escape
nuclear entry

process of intracellular transport and processing is not fully understood yet!

43
Q

AAV and the immune system

A

regarded as low-immunogenig
sensed by TLR
activates innate immune system
neutralized by pre-existing neutralizing antibodies
active immune response (T-cell based) usually only against transgene product and not against the capsid

it was seen that AAVs usually do not efficiently transduce antigen-presenting cells (APCs)

44
Q

points to consider on which AAV to chose

A

1) target tissue/cell type
2) safety profile of delivered transgene product
3) choice of delivery route
4) choice of tissue-sepecific or constitutive promotors

45
Q

humoral immune response to AAV

A

AAV peptides get uptaken by APCs
presented by MHC-II
activates B-cells and CD4+ T-cells
plasma cell and memory cell development is induced
AAV specific antibodies are produced
either neutralizing or non-neutralizing antibodies

46
Q

nAbs

A

neutralizing antibodies

promote killing of infected cell through T-cell answer

47
Q

non-nAbs

A

non-neutralizing antibodies

are thought to opsonize AAV particles and promote their degradation through the spleen

48
Q

hypothesis regarding point of uncoating of AAV

A

1) following endosomal escape, capsid breakdown and uncoating occurs after subsequent nuclear translocation
2) cytosolic ubiquitination of the intact capsid can occur during transport to the nucleus

49
Q

AAV production systems

A

1) tripple transfection of HEK293 cells (transgene, AAV helper, pXX)
in adherent or suspension cultures -> 10E+05 GC/cell and 10E+14 GC/L
2) producer cell lines where one or more genetic component has already been integrated into mammalian/insect cells
2a) co-infection of BHK cells with two replication-deficient HSVs encoding for the transgene and rep/cap
2b) stable producer HeLa cells carrying transgene and rep/cap (problem: needs to infected with wtAAV for production -> safety concern!)
3) insect cell lines
Sf9 cells in combination with baculovirus infection (usuage of two or more viruses which carry Rep, Cap, transgene in single viruses; transgene might also be stably integrated into Sf9 cells) -> 10E+05 GC/cells and 10E+15 GC/L of production yield

50
Q

AAV vector purification methods

A

iodixanol densitiy gradient ultracentrifugation
caesiumchloried densitiy gradent ultracentrifugatoin
affinity chromatography (employed by industry)

51
Q

Glybera

A

alipogen tiparvovec

first EMA-approved AAV based product

52
Q

voretigene neparvovec-RPE65

A

most advanced current clinical trial (Phase III studies completed)
sponsored by Spark Therapeutics
utilizes local AAV2 injection into the eye
treatment for inherited retinal diseases

53
Q

integration of AAV

A

wtAAV can integrate its genome through the Rep proteins and ITR’s
AAV2 prefers the AAVS1 locus
large Rep proteins bind to RBS in ITR’s, unwind the DNA and produce a single-stranded nick in the trs

an RBS homoloug present in the p5 promotor can act as RBS in the absence of ITR’s

around 10-20% of cells infected with AAV2 displayed AAV integration within a 4kb region of AAVS1
on selected cells the integration rate increased to 80%

problems of analysis of integration clones: PCR methods face the problem of the extremely GC-rich ITR regions

besides AAVS1 numerous RBS and trs have been found in the human genome
AAVS2 located on chromosome 5p13.3
AAVS3 located on chromosome 3p24.3

54
Q

AAVS1

A

first identified preferential integration site of AAV2
located in the human chromosome 19
safe harbour region
contains homolouges sequences to RBS and trs elements
AAV can integrate at variable distances from the RBS and sequence rearrangment were frequently found in AAV-chromosom junctions

55
Q

LAM-PCR

A

linear amplification mediated PCR

used for integration analysis of retroviruses

56
Q

LM-PCR

A

ligation mediated PCR
employed for HIV and MLV integrations successfully before
relies on a first LTR specific primer
a linker is ligated to the first PCR strand that ends at a chason restriction site within the unknow chromosomal sequence
a primer complementary to this linker ensures second-strand synthesis
retrovirus-chromosom junctions are amplifiied using a combination out of linker- and LTR-specific primers

57
Q

LSM-PCR

A

adapted LM-PCR from Heilbronn et al = linker-selection-mediated PCR

1)genomic DNA of cells is cleaved with restriction enzymes
2)sequences were amplified with one primer a unique AAV-sequence and the second primer binds to the linker attached to the unknown chromosomal site
structure of the linker forces PCR to initiate amplification within AAV genome
biotin-tag was attached to the linker for enrichment

Adeno associated virus (2 decks)

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