190 immune response and growth factor formation after direct intra articular injection of AAV2 or AAV5 IGF i in an equine model

190 Immune Response and Growth Factor Formation After Direct Intra Articular Injection of AAV2 or AAV5 IGF I in an Equine Model Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The Amer[.]

Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy

S72

IMMUNE RESPONSES TO CELL AND GENE THERAPY

Figure 1 Oncolytic virus can replicate in cancer cells increased

several ten thousand folds, the inserted gene can also be replicated

with increase of several ten thousand folds

The effect of CTGVT is not 1+1=2 but equal to 50-100 (the fi gure

is omitted) Therefore, they both should go through to carried out

the CTGVT stategy The CTGVT (OV-gene) is a revolution of gene

therapy and oncolytic viral therapy Furthermore, we still made the

modifi cation of the CTGVT by the use of two gene strategy, the

CTGVT-DG (double gene), bucause two genes may have synergetic or

compensative effect between them By the of CTGVT-DG strategy, all

the xenograft tumor of many cancers could be essentially completely

eradicated

Figure 2 Complete eradication of the colorectal cancer by

ZD55-TRAIL+ZD55-IL-24 due to their synergetic effect

The CTGVT-DG is the top antitumor effect of CTGVT In the past

years, we always used the OncoAd (OV from adenovirus) as vector,

however, the OncoPox (OV from vaccinia virus of Poxvirus) has many

superiority by its replication in cytoplam and never integration to

chromsome of genome, OncoPox will never induce cancer and is the

fi rst virus to be i.v injected to human Thus, from 2011, we developed

the strategy of the combination of CTGVT-DG and OncoPox, then

double targeting OncoPox by double deletion of TK and B18R of

vicinia virus (Tk-, B18R- OncoPox, vvDD) and harboaring double

genes (vvDD-gene1-gene2) was innovated The antitumor effect of

our vvDD-gene1-gene2 will have much much better antitumor effect

than that of the current most antitumor drugs, the OncoPox-GM-CSF

which published a paper in Nature in 2011 and Nat Med in 2013, and

also much better antitumor effect than that of OncoHSV-GM-CSF

(OV from Herpe Sinplox Virus) which Amgen paid 1 billion USD

to BioVex The higher atntiumor effect of our constructed drugs is

due to that they have double genes for cancer theapy A patent of

OncoPox-gene1-gene2 is in application

Immune Responses to Cell and Gene Therapy

190 Immune Response and Growth Factor Formation After Direct Intra-Articular Injection of AAV2- or AAV5-IGF-I in an Equine Model

Kyla Ortved,1 Bettina Wagner,1 Alan Nixon.1

1 Cornell Unversity, Ithaca, NY.

Intra-articular viral vectors expressing therapeutic transgenes are potential treatments for OA AAV is a common vector used for gene transfer but concerns regarding neutralizing antibodies (NAb) and

T cell responses have been expressed The immune response to IA vectors remains largely unknown We hypothesized that AAV2- or AAV5-IGF-I would result in IGF-I formation but would induce NAbs and cytotoxic T cell responses 12 healthy horses were assigned to treatment (AAV2 or AAV5) or control (saline) groups Middle carpal (MC) joints were injected with 5e11vg in 3ml of DPBS Each horse had clinical evaluations and arthrocentesis on days 0,4, 7, 14, 28 and

56 Synovial fl uid was analyzed for total protein (TP) and nucleated cell counts (NCC), NAbs, cytokines including IL-10, IL-6, IFN-γ, sCD14, and CCL2, and IGF-I concentration Serum was evaluated for cytokines and NAbs Blood was collected prior to injection, at day 14 and day 56 PBMCs were isolated using Ficoll centrifugation Cells were stimulated with PMA/iono, media, AAV2 or AAV5 At 48h cells were harvested, fi xed, permeabilized, and stained for CD4 or CD8 and for IFN-γ, IL-4 or IL-10 Cells were analyzed using a fl ow cytometer with fl uorescence gates set according to isotype control staining Cells were also plated in 96-well plates Supernatant was collected at 48h for cytokine secretion analysis Injection of AAV2- or AAV5-IGF-I did not induce greater articular infl ammation compared

to saline TP levels were slightly increased in AAV5-IGF-I joints at days 14 and 28; however, these increases were not signifi cant (fi g 1) NCCs were signifi cantly increased in AAV5-IGF-I joints on day

14 (3375 ± 957.73 cells/mL; p<0.0001) only (fi g 1)

IGF-I concentrations from AAV5-IGF-I joints were signifi cantly increased over saline at days 14, 28 and 56 IGF-I mRNA expression was 25 times higher in synovial membrane tissue from AAV5-IGF-I joints than saline (fi g 2)

After restimulation of PBMCs with AAV2 or AAV5, horses injected with either virus did not have increased IFNγ+ lymphocytes in either CD4+ or CD8+ populations indicat-ing lack of cellular immune response All AAV5 horses had increased NAb titers in serum and synovial fl uid whereas

Molecular Therapy Volume 22, Supplement 1, May 2014

IMMUNE RESPONSES TO CELL AND GENE THERAPY

2 AAV2 horses were non-responders with no detectable

increases Injection of AAV2- or AAV5-IGF-I into the MC

joint of horses did not induce a clinically detectable infl

am-matory response IGF-I concentrations in the synovial fl uid

of AAV2- and AAV5-IGF-I injected joints increased over

control No cellular immune response was detected although

a humoral response was noted especially in AAV5 treated

horses.

191 Treg Migration Into the Bone Marrow and

Spleen Mitigates Rapid Reversal of Pre-Existing

Anti hFIX Antibodies Following AAV hF9 Liver

Gene Transfer

David M Markusic,1 Roland Herzog.1

1 Pediatrics, University of Florida, Gainesville, FL.

Previously we have demonstrated robust reversal of pre-existing

anti human factor IX (hFIX) antibodies, inhibitors, following

AAV8-hF9 liver gene transfer This reversal was dependent on the activation

and maintenance of hFIX specifi c regulatory T cells (Treg) The

goal of the following studies was to further defi ne a mechanism and

timeline of events following liver gene transfer In these studies we

worked with a hemophilia B murine model on a Balb/c background

(Balb/c F9-/-) In contrast to C3H/HeJ F9-/- mice, we and others have

inhibitory antibodies to recombinant hFIX protein therapy and do not

develop hypersensitive reactions Mice in this study were immunized

with hFIX protein in adjuvant, which has proven a more reliable

method to induce hFIX antibodies In an initial study we induced

hFIX antibodies in two groups (n=8/group) of Balb/c F9-/- mice and

then gave AAV8-hF9 liver gene transfer to one group and followed

inhibitor levels over time Strikingly we observed a complete loss of

inhibitors two weeks following liver gene transfer that was maintained

following a second hFIX adjuvant challenge The rapid loss of

inhibitors coupled with our previous data suggested that there was

active elimination of hFIX antibody secreting B cells (plasma cells)

and suppression of hFIX memory B cells Long-lived plasma cells

(PC) generate the majority of high affi nity antibodies and primarily

reside in survival niches found in bone marrow (BM) and to a lesser

extent in the spleen Therefore we hypothesized that elimination of

hFIX secreting PC in the BM, within two weeks following liver gene

transfer, may involve Tregs To examine this we generated two groups

of Balb/c F9-/- mice (n=3/group) immunized with hFIX protein in

adjuvant Following confi rmation of hFIX antibodies one group of 3

mice received AAV8 hF9 liver gene transfer Twelve days following

liver gene transfer we collected spleen and bone marrow cells for

analysis Bone marrow cells were stained for antibodies to mark B

and FoxP3) BM cells from mice given AAV8-hF9 vector stained

higher for Treg and lower for double positive B cells and PC when

compared against immunized control mice In a parallel study we

looked at the baseline viability of B cells and PC in MACS enriched

splenic B cells following three day in vitro culture Under our culture

conditions we observed a higher proportion of dead PC in vector

treated compared to control cells Given that the PC were cultured

in the absence of hFIX specifi c Treg suggests that the reduction in

live PC is a result of previous elimination by Treg prior to collection

Ongoing experiments are planned to examine the impact of hFIX

Treg and B cell/PC co-culture on viability and antibody secretion

Based on our initial experiment we plan to look at earlier time points

(3 and 7 days post gene transfer) to get a better understanding on

Treg induction and migration kinetics Our preliminary data support

a model of rapid induction and migration of hFIX specifi c Treg to

BM and spleen to eliminate anti-hFIX secreting PC

192 Neutralizing Antibodies Against AAV Capsids in Patients with Mut Methylmalonic Acidemia (MMA)

Jennifer L Sloan,1 Irini Manoli,1 Elizabeth Harrington,1 Randy J Chandler,1 Mark Schneider,2 Roberto Calcedo,2 James M Wilson,2 Charles P Venditti.1

1 Organic Acid Research Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD; 2 Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA.

INTRODUCTION: Methylmalonic acidemia (MMA) is a severe autosomal recessive inborn error of metabolism characterized

by lethal metabolic instability, multiorgan pathology, and a poor prognosis for long-term survival The disorder exhibits genetic heterogeneity and is most commonly caused by complete (mut0) or partial (mut–) defi ciency of the enzyme methylmalonyl-CoA mutase (MUT) Elective liver (LT), kidney (KT) or combined liver kidney transplantation (LKT) are used to stabilize the most severely affected patients We have demonstrated that systemic AAV gene delivery is highly effective in mouse models of MMA, which has encouraged future translation to patients We sought to survey AAV neutralizing antibody (NAb) titers against serotypes 2, 8, and 9 in a group of well-characterized MMA patients, anticipating that this data could inform the selection of an optimal serotype to use in a gene therapy clinical trial

METHODS: Participants and biospecimens were accrued via NIH study “Clinical and Basic Investigations of Methylmalonic Acidemia and Related Disorders” (clinicaltrials.gov identifi er: NCT00078078) NAb titers were measured using an AAV-CMV-lacZ reporter to infect Huh7 cells after preincubation with serial dilutions of patient plasma

to measure NAb titers as low as 1:5, as described (Calcedo R, et al.,

J Infect Dis 2009 199:381)

RESULTS: 46 samples from 42 patients with mut MMA between the ages of 2-30 years (ages 2-5, n=13; ages 6-11, n= 15; ages 12-20, n=7; age 20+, n= 11) were fi rst studied to examine AAV8 and AAV9 Nab titers These patients included 9 mut- and 33 mut0 patients; 10 had received a KT, LT or LKT The total seroprevalence of Nabs against AAV8 (n=9) or AAV9 (n=10) was 19% and 21% respectively with 90% co-seropositive Those with detectable Nab antibody titers were typically older than 20 years (n=7), mut- (n=6) or had received solid organ transplantation (n=7) Among the 23 mut0 patients who had not been transplanted, only the oldest individual, age 24, was weakly co-seropositive with 1:5 NAb titers Next, we measured AAV2 NAbs in 20 patients, including all those seropositive against AAV 8 and 9, as well as a subset of clinically severe or older mut0 patients, aged 2-28 In this group, 6 patients (30%) were seropositive, 5 were tri-seropositive, of whom 4 had received organ transplants

CONCLUSIONS: The incidence of NAb titers against common AAV capsids in patients with MMA has encouraging implications for systemic gene delivery Those with more severe clinical and enzymatic phenotypes who have not received KT, LT or LKT - i.e the group of patients with the greatest need for gene therapy - largely lack NAbs against AAV 2, 8 and 9 capsids Whether this refl ects a manifestation of impaired humoral immunity conferred by the disease state remains unknown