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AAV CAPSID EVOLUTION
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Recombinant adeno-associated virus (AAV) is a highly popular gene delivery
vector for a wide range of gene therapy and vaccine applications, thanks to its
broad tropism, prolonged transgene expression, non-pathogenicity, and low
immunogenicity.
However, several problems with existing AAV serotypes limit their therapeutic
potential. First, while available serotypes offer a variety of tropisms to
choose from, many clinical applications require tissue-specificities that fall
outside of their coverage. Second, even if a tissue of therapeutic interest is
covered by the tropism of one or more serotypes, the efficiency of gene delivery
may be too low and there may also be undesirable tropism for off-target tissues.
Third, pre-existing neutralizing antibodies against many AAV serotypes can block
their efficient delivery initially or with repeated drug administration. Lastly,
some serotypes are inherently difficult to manufacture at high titer, purity and
stability. To overcome these limitations, AAV capsid engineering has been a
critical area of research that has significantly accelerated the development of
novel AAV variants with improved features.
Directed evolution is a widely used high-throughput approach to engineer
enhanced biomolecules. It mimics the process of natural selection through
repeated genetic diversification and selection. Directed evolution of AAV capsid
is performed by mutating the wildtype AAV capsid gene to generate highly diverse
AAV capsid libraries, which are then screened to identify novel capsid variants
with improved properties. Since directed evolution does not require prior
knowledge of the structure-function relationship of proteins, it is often
preferred over rational design for AAV capsid engineering.
SERVICE HIGHLIGHTS
* Full-service platform: VectorBuilder is the world’s only company capable of
providing one-stop solution for all your AAV preclinical and clinical CRO and
CDMO needs. Our AAV services encompass vector design and optimization, vector
cloning, library construction, virus packaging, capsid evolution and targeted
engineering, AAV biodistribution profiling, and GMP manufacturing.
* High complexity capsid library construction via versatile approaches: With
our extensive expertise in constructing pooled libraries, we can help you
design and custom build highly diverse capsid libraries via any mutagenesis
approach or combinatorial approach.
* High titer capsid library virus packaging: We can do either one-step or
two-step virus packaging of the capsid library to ensure each viral particle
to contain corresponding capsid variant in its genome and to achieve high
virus titer.
* In vivo screening in multiple species including nonhuman primate (NHP):
Besides in vitro screening capabilities, we offer a wide range of species on
our in vivo screening platform, such as mouse, rat, and importantly, two
nonhuman primate species: crab-eating macaque (Macaca fascicularis; a.k.a.
cynomolgus monkey) and rhesus macaque (Macaca Mulatta). In vivo screenings
are carried out in AAALAC-accredited facilities by highly trained
professionals.
* Full technical support: Our highly experienced scientists can provide
comprehensive technical support covering every aspect of your AAV capsid
project, ranging from library construction to in vivo screening, from library
design to NGS analysis.
WORKFLOW OF CAPSID EVOLUTION AND SCREENING
The first and the most critical step in the entire workflow of AAV capsid
evolution is the generation of a highly diverse AAV capsid library in which each
plasmid carries a chimeric AAV genome consisting of a rep gene and a capsid gene
variant. The capsid gene variants can be efficiently generated using various
approaches, such as error-prone PCR, random peptide display, DNA family
shuffling, or in silico design. The capsid library is then packaged into viral
particles, and each viral particle harbors corresponding capsid variant in its
genome. The viral library is subsequently subjected to a screening process to
test the ability of chimeric AAVs: 1) to efficiently transduce target cells
within specific tissues or organs, or 2) to bind to cell type-specific receptors
at high affinity, or 3) to evade neutralizing antibodies. Viral genomes passed
screening are recovered from target cells and made into a smaller library for a
second round of screening. Several rounds of screening are usually performed to
enrich high confidence hits. The resulting hits are then validated and
characterized to identify novel AAV capsid variants with enhanced properties
(Figure 1).
Figure 1. Typical workflow for screening novel AAV capsids by directed
evolution.
CAPSID LIBRARY CONSTRUCTION
The strategies described below are commonly used for creating diverse AAV capsid
variants. Then, capsid library for screening is constructed by massive parallel
cloning of the capsid variants into AAV vector to form chimeric AAV genomes
(each consisting of a rep gene and a capsid gene variant).
Error-prone PCR
Error-prone PCR is the most straightforward approach for developing highly
variable AAV capsid libraries which involves the modification of standard PCR
methods to mutagenize the AAV capsid gene. More specifically, error-prone PCR
employs a combination of various sub-optimal PCR conditions including low
fidelity polymerases, longer extension times, higher Mg2+ concentrations,
addition of Mn2+, and varying dNTP concentrations for introducing random point
mutations into the AAV capsid gene.
Random peptide display
Random peptide display involves the insertion of random peptide sequences of
usually 7 to 9 amino acids into specific sites of the AAV capsid with the aim of
altering the natural cellular interactions of the virus and retargeting it to
specific cell receptors. The peptides are typically inserted into locations of
the AAV capsid that facilitate surface exposure of the peptide and are also
critical for virus-host interactions. For example, between position 587 and 588
(within the variable region VRIII) of the AAV2 capsid is a preferred insertion
site for most AAV2-based peptide display libraries since insertion of peptides
in that region abolishes the heparan sulfate proteoglycan (HSPG, the primary
AAV2 receptor) motif of AAV2 and enables the displayed peptides to interact
efficiently with cell surface molecules.
DNA family shuffling
DNA family shuffling is a highly efficient approach for generating chimeric AAV
capsids by molecular interbreeding of parental capsid genes derived from
different AAV serotypes. To accomplish this, the parental capsid genes of
various AAV serotypes are fragmented, followed by their reassembly into novel
full-length capsid variants by primer-less PCR which recombines them based on
partial sequence homology. As an alternative strategy, high complexity libraries
can also be created by synthetic shuffling which combines rational design
(modifying the capsid based on prior knowledge of AAV biology) with directed
evolution. In this approach, capsid locations suitable for mutagenesis are first
identified and evaluated based on a detailed structural and sequence analysis of
naturally occurring AAV serotypes. Fragments containing mutations are
synthesized and assembled to form full-length novel capsid variants.
In silico design
In silico design of AAV capsid libraries utilizes a variety of approaches for
computational prediction of capsid variant sequences with the potential to
contribute to enhanced AAV performance. One commonly used approach is ancestral
reconstruction which involves in silico designing of a putative ancestral AAV
library followed by its experimental validation for identifying highly potent
ancestral capsid sequences with improved tropism. The rationale behind this
approach is that evolutionary AAV intermediates that emerged by surviving the
process of natural selection are highly likely to possess unique properties
while maintaining virus structure and function. Machine learning is another
commonly used in silico design approach that applies computational algorithms to
predict the chances of viable virus production from hypothetical capsid
variants. Machine learning algorithms heavily rely on available input data to
learn structure-function relationships of proteins and apply that to predict the
outcome of complex physiological processes such as viral capsid assembly.
VIRUS PACKAGING OF CAPSID LIBRARY
Virus packaging of capsid library can be accomplished by either one- or two-step
process as described below:
One-step packaging of capsid library
The conventional approach for packaging AAV capsid libraries utilizes a one-step
process in which packaging cells are co-transfected with capsid library and an
adenoviral helper plasmid. While this approach is widely used, it does come with
the disadvantages of cross-packaging (generation of AAV particles with
mismatched capsid variant genome and capsid) and capsid mosaicism (generation of
AAV particles with mosaic capsids arising from capsid proteins derived from
different genomes). To overcome these challenges, it is recommended that the
packaging cells are transfected at a very low plasmid library to cell ratio to
ensure the uptake of at most one single library plasmid per cell.
Two-step packaging of capsid library
In a two-step packaging approach, the capsid library is first co-transfected
into packaging cells along with a helper plasmid encoding the WT capsid gene but
lacking the viral ITRs. This results in the production of AAV particles with
mosaic capsids that are partially made up of the WT capsid and are referred to
as AAV transfer shuttles. The AAV transfer shuttles are then introduced into
packaging cells at a low MOI for achieving at most one viral particle per cell,
followed by superinfection of the packaging cells with adenovirus which
ultimately results in the generation of high-titer viral capsid library.
CAPSID SCREENING IN VITRO AND IN VIVO
The viral capsid library is usually subjected to several rounds of screening in
vitro or in vivo, based on screening purpose, to select for chimeric AAVs with
desirable properties:
In vitro selection
Utilizing established cells lines for AAV capsid library selection is a widely
used approach, particularly for identifying AAV variants with altered receptor
targeting abilities. Although in vitro selection of AAV libraries is fast and
technically simple, it does present some challenges. Firstly, vectors optimized
for high transduction efficiency in vitro may not be able recapitulate the same
efficiency when used in vivo. Secondly, AAV vectors demonstrating high degree of
target cell specificity in vitro might transduce non-target tissues when
translated in vivo. Another strategy for in vitro selection of AAV libraries
involves subjecting the library to potent serum from immunized animals prior to
adding it to target cells, specifically for identifying variants with immune
evasive properties. However, immune response of AAV variants may vary when
translated in vivo due to various factors (e.g. immune recognition of the same
AAV vector may vary when delivered via different routes).
In vivo selection
In vivo animal models offer a more reliable platform for screening AAV
libraries, particularly for identifying AAV variants capable of transducing
delicate cell types that cannot be grown in culture or AAV variants capable of
transducing a specific cell type with a complex tissue structure. In vivo
selection also helps to identify any potential off-target effects associated
with an AAV variant. While both mouse and NHPs are widely used for in vivo
selection of AAV libraries, NHP models represent the most clinically relevant
platform for screening improved AAV vectors due to their high degree of
similarity to humans.
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