﻿First, I want to thank the meeting organizer for giving me the opportunity to talk
at this very exciting conference. Also thank the audience for listening to my talk.
Here are my disclosures. In AAV mediated in vivo gene transfer, there are two key immunological
components. First, is the capsid. How AAV dresses matters. It determines tissue tropism and
transduction efficiency. It directs intracellular trafficking. It delivers gene payload to the
nucleus. Importantly, it also dictates host immune responses. And today, I have no time to
talk about host immunity to AAV capsid. However, I would like to summarize it as the following.
First the B cell immunity to the capsid. Preexisting neutralizing antibody prevents
effective gene transfer, narrowing eligible patient population. Vector capsid induced by
neutralizing antibody induce the neutralizing antibody prevent redosing of the same vector.
Capsid may induce dose limiting complement activation, platelet reduction, and renal failure.
Mitigation strategy includes plasmapheresis and capsid specific IgG absorption,
alternative serotypes or capsid engineering for immune escaping,
IgG cleaving, endopeptidase digestion, or transient immune suppression regimens.
In terms of B cell immunity to capsid, which is potentially caused by memory T cell activation,
cross presentation, bystander activation, and other mechanisms leading to transaminitis.
Mitigation strategy includes corticosteroids, additional transient immune suppression regimens.
The second component is AAV vector genome, which is
therapeutic payload carrier. Mediates gene therapeutic benefits, achieves long term stay. It
also triggers innate pattern recognition receptor engagement and adaptive transgene immunity.
I wanted to give an overview on AAV innate immune interactions. When the AAV enter the body,
first they encounter preexisting neutralizing antibody. And then get engaged with TLR2 receptor
on the antigen presenting cell surface and trigger the capsid sensing. And second,
could internalize into the endosome engage with a TLR9 and MLD88 and trigger being a sensing
involved with cGAS and STING pathway, as well as the M2 pathway as shown in here. And, finally,
AAV could potentially trigger double strand and single strand RNA sensing.
Many years ago, pioneer work by Dr. Jim Wilson demonstrated that CpG depleted
AAV evades immune response that use TLR9 lockout show that diminished transgene and capsid specific
T cells and prolongs transduction. They also use the same lockout to show the reduced inflammatory
cytokines and chemokines response and CpG depleted AAV invades immune response. They use TLR9 lockout
show that diminished transgene and capsid specific T cell and prolongs transduction. They also use
the same lockout show the reduced inflammatory cytokines and chemokines response at early stage
of AAV transduction. When deplete, CpG motifs they found that transgene cDNA depletion of the
CpG motif reduces transgene and capsid specific T cells and stabilize muscle transduction.
And in terms of implication, clinical implication level with AAV gene sensing,
recently Frasier Wright did a retro perspective study and suggests that stability of AAV
hemophilia B clinical gene therapy is negatively correlated with number of CpG motifs in transgene
cDNA. In a more recent study with hemophilia B, gene therapy suggests the same. And importantly,
such a immune response to AAV is not always steroids responsive.
And Frasier also proposed that computational approach CpG depleted AAV invades immune
response. They use TLR9 lockout showed that diminished transgene and capsid specific T
cell and prolongs transduction. They also use the same lockout showed a reduced inflammatory
cytokines and chemokines response at the early stage of AAV for animals. TLRs and STING pathways,
which is demonstrated by transduction in different local animals.
AAV and AIM2 lockouts increased AAV transduction in early and late time points but has no impact
on anti drug antibody IgG response. Here as showing by AAV1 Ova muscle transduction,
as well by AAV9 A1AT/IV injection. So, in summary, engineering cDNA for
sustained strong expression of the transgene require us to consider two important factors. And
if we reduce CpG motifs, we could potentially increase stability of transgene expression.
But if we optimize codon usage, we should be able to enhance
expression level. Our job is to balance the two factors, accomplish stable high expression.
Recently, George Church and Ying Kai Chan come up a very elegant design
to inhibit a DNA sensing. They incorporated TLR9 inhibitory sequence in AAV genome.
And then they found this sequence can enhance AAV muscle transduction and suppress CD8
T cell response. They also found that this sequence can enhance AAV transduction in eyes
after intravitreal delivery. Our lab also tested this system and found this sequence can suppress
inflammatory cytokines as early as three hours after AAV mediated Ova muscle injection.
And so, now I am going to switch the gear to adaptive transgene immunity. And in this case,
we found that AAV specific antigen presentation by professional antigen presenting cells is a main
cause for adaptive transgene immunity. When AAV inadvertently transfuse antigen presenting cells,
the capsid will be processed as capsid peptides. And when enter the nucleus
and transcribe and express the transgene messenger RNA and the protein, then this messenger this
protein will be processed and present together with some capsid peptides as class I molecule
trigger the CD8 T cell response. Alternatively, those capsid peptides can be also presented
as class II molecule trigger CD4 T cell response. So, since the antigen presentation of the
transgene is the major cause of adaptive immunity, so, the strategy to reduce transgene immunity
should be focused on detargeting from antigen presenting cells. The first strategies use
T cell specific promoter for transcription of detargeting. In this case, if we use a viscus
promotor it can transduce a perfect tissue and generate a beautiful expression. But same time,
this vector can also if inadvertently transduce the antigen presenting cells,
could also present the transgene as a class I molecule trigger CD8 T cell response.
However, if we switch the promotor to a tissue specific such as liver specific after injection,
we won't get a liver specific expression, but when this AAV transduce antigen presenting cells,
since this promotor will not work efficiently in these cells, it will generate less or reduce
antigen presentation. And then as showing by this ELIspot assay showing tremendous reduction by
TBG promotor issues of ELIspot to (inaudible) transgene. This also can be used to express highly
immunogenic antigen ovalbumin and as you can see, TBG significantly enhanced in transduction
as compared to viscous promotor. So, the second strategy is to use
cell specific endogenous microRNA for posttranscriptional detargeting from antigen
presenting cells. In this case, you incorporate microRNA binding site that is specific to antigen
presenting cells, specifically microRNA and then when you get into other tissues, such as
brain and muscle, since none of those APC specific microRNA you get a beautiful expression.
However, when this AAV transduce antigen presenting cells and those cells they have
endogenous expression of the specific microRNA form RNA induced complex. And then will A,
be transduced into those cells, and express the messenger RNA of the transgene will be
clipped by those rest. And then you will have reduced antigen presentation on the surface.
To test this principle in non human primates, my collaborator Ron Desrosiers put those
micro binding sites into AAV expressing broadly neutralizing antibody to HIV virus.
When he put this virus into monkey muscle, there is two possibilities. One possibility if you have
anti drug antibody response, you will have neutralized vector of the antibody. And
if you have those micro incorporated and then hopefully you can reduce an anti drug antibody
response and you have successful expression of the broadly neutralizing antibody kill HIV virus.
And then he put into a monkey model GFP infection a single injection can reduce the GFP level to
undetectable for at least three years. The bottom line is that Ron created a Miami monkey that
is functional cured by micromanaged AAV vector expressing broad neutralizing antibody to HIV.
So, encouraged by this data, my colleague, Manish Muhuri, continued screening his screening
for a more potent APC specific microRNAs. He used an in vitro assay use ovalumin as
a sensitive reporter gene. And he once searched for the microRNA as high expression in myoblasts
and low expression in antigen presenting cells. He used microRNA 142 binding site as a reference then
he found two binding sites that have strong inhibition to APC antigen presentation.
And then he put those binding sites in combination with our result, microRNA 142 binding site and
inject into muscle expressing ovalbumin. As you see here that only the AAV without the microRNA
binding site have no ovalbumin expression or other binding site vector as strong and high
level expression. And then on the upper set, in the upper set that when you look at anti OVA
IgG, only the vector without microRNA binding site has strong anti IgG response IgG response
but not microRNA binding site adapter. When the endocanna (phonetic) infiltrates
in histology in the muscle tissue, a dramatic reduction of infiltrates in the microRNA binding
site as compared to AAV without microRNA detargeting and quantified as this progress.
And then with STING based CD8 and (inaudible) B, the same pattern was showing here.
And on the other hand, when you quantify persistent vector genome you will see
microRNA binding site and remarkably increased persistence of the vector genome as compared to
without binding site in the muscle. And also, STING based F480 markers also show the similar
inhibition pattern with the microRNA binding sites.
And to understand the mechanism of how this works, Manush performed this study and he found that
novel APC detargeting microRNA binding sites blunt ADA induction by altering APCs
and the T cell to a transgene specific suppressive phenotypes as showing by macrophages, by dendritic
cells, CD4+ T cells, ovalbumin specific CD8+ T cells, CD4 T cells, and CD4 and CD8 memory T
cells, and CD80 positive and CD80 positive 86 positive dendritic cells as well as TH1 and TH17
helper cells from their splenocytes as well as relative cytokine expression.
Thank you very much. I would like to thank my colleagues in the group
and the other non members, and research sponsors to support our research. Thank you.