Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy S184 Adenovirus and Other DNA Virus Vectors Binding to Adenovirus Hexon Reveal
Trang 1Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
S184
Adenovirus and Other DNA Virus Vectors
Binding to Adenovirus Hexon Revealed a Binding
Pocket for Coagulation Factor GLA Domain That
Defi nes the Infectivity of Adenovirus-Coagulation
Factor Complexes
Eric E Irons,1 Justin W Flatt,2 Konstantin Doronin,3 Tara L Fox,2
Mauro Acchione,1 Phoebe L Stewart,2 Dmitry M Shayakhmetov.1
1 Department of Medicine, University of Washington, Seattle, WA;
2 Department of Pharmacology, Case Western Reserve University,
Cleveland, OH; 3 Department of Molecular Microbiology and
Immunology, St Louis University, St Louis, MO.
Adenoviruses (Ad) are promising vectors for therapeutic
interventions in humans However, when injected into the
bloodstream, Ad vectors can bind several vitamin K-dependent blood
coagulation factors, which contribute to virus sequestration in the liver
by facilitating transduction of hepatocytes Although both coagulation
factors FVII and FX bind HAd5 hexon with very high affi nity, only
FX appears to play a role in mediating Ad hepatocyte transduction
in vivo To understand the discrepancy between effi cacy of FVII
binding to the virus and its poor capacity at supporting Ad cell entry,
we analyzed the HAdv5-FVII complex using high-resolution
cryo-electron microscopy, followed by molecular dynamics fl exible fi tting
(MDFF) simulations The results indicate that the FVII GLA-domain
sits within a surface-exposed hexon trimer depression in a different
orientation than found for FX, although several of the same hexon
residues that are involved in mediating FX binding also mediate FVII
binding to hexon (T423-E424-T425) Furthermore, we found that
when bound to the WT hexon, two proximal FVII molecules interact
via their serine protease (SP) domains and bury potential heparin
sulfate proteoglycan (HSPG) receptor binding residues within the
dimer interface While FVII binds WT hexon with Kd=3nM, E424A
or T423G-E424A amino acid substitutions reduced FVII binding
10-fold (Kd=30 nM) Surprisingly, despite the equal binding affi nity
of FVII for E424A and T423G-E424A hexon mutants, the vector
possessing a two-amino acid substitution demonstrated superior cell
transduction effi cacy in the presence of FVII, when compared to either
the WT or E424A mutant MDFF simulations of FVII binding to
E424A and T423G-E424A hexon mutants revealed altered interfaces
between hexon and FVII that could affect the orientation of FVII
and, thus, the effi cacy of SP domain dimer formation Because FX
interaction with hexon does not lead to the formation of SP domain
dimers that occlude HSPG cell receptor interacting sites, our data
suggests that upon FVII binding to T423G-E424A, but not to the
WT or E424A mutant, SP domain dimer formation is either ablated
or ineffi cient, leading to highly effective virus-mediated gene transfer
in the presence of FVII Thus, our structural, computational, and
biochemical analyses revealed important roles for specifi c hexon
amino acids that lead to different binding orientations for FX and FVII
Gla domains, which lead to a drastic variation in cell-transduction
effi cacy of the resultant coagulation factor-Ad vector complexes Our
study provides important information on the dynamic nature of the
coagulation factor-Ad hexon interaction and may aid in the design
of novel safe and effective Ad vectors, amenable to cell type-specifi c
targeting after intravascular delivery
478 Identifi cation of a Negative Regulator of TRPV1 Via an HSV Based cDNA Library Screen
Bonnie L Reinhart,1 Asaff Harel,1 MingDi Zhang,1 James R Goss,1 William F Goins,1 Justus B Cohen,1 Joseph C Glorioso.1
1 Department of Microbiology and Molecular Genetics, University
of Pittsburgh School of Medicine, Pittsburgh, PA.
Chronic pain occurs when normal control of acute pain signaling
is lost, resulting in reduced stimulus thresholds and hypersensitive sensory neurons The transient receptor potential vanilloid channel (TRPV1) is a pro-nociceptive cation channel that is activated in response to noxious stimuli such as capsaicin, heat, or low pH TRPV1 is functionally up-regulated in pathophysiological states such
as painful diabetic neuropathy and chronic infl ammation We have developed an HSV replication-based selection strategy to identify novel proteins that negatively regulate TRPV1 Over-expression of TRPV1 from an HSV vector causes calcium infl ux and cell death
in the presence of capsaicin, thereby inhibiting virus growth Co-expression of a cDNA library and TRPV1 from the same backbone should allow replication and selection of only those vectors that express negative modulators of TRPV1’s response to capsaicin To create this library, we developed an HSV vector (2xTRPV1) that contained two essential modifi cations: 1) a Gateway expression cassette to facilitate insertion of a cDNA library under control of the CMV promoter, and 2) duplicate copies of the TRPV1 gene, under control of the viral TK promoter, to reduce the selection of TRPV1-defective mutants As anticipated, the 2xTRPV1 vector failed to replicate in the presence of capsaicin In contrast, a control vector that co-expressed the two TRPV1 genes and TRPV1-poreless,
a dominant-negative mutant of TRPV1, was able to replicate in the presence of capsaicin A cDNA library derived from PC12 cells was introduced into the 2xTRPV1 vector backbone and screened for TRPV1 antagonists Viruses that escaped capsaicin-induced cell death were isolated and the corresponding cDNAs were sequenced Candidate cDNAs were introduced into a fresh selection vector and retested for TRPV1 inhibition One candidate gene, PP1, rescued virus growth in the presence of capsaicin to a level similar to that
of TRPV1-poreless PP1 expressed from a replication-defective vector reduced capsaicin-induced calcium infl ux in rat primary dorsal root ganglion (DRG) neurons and, similar to a TRPV1-poreless vector, decreased sensitivity to thermal pain in Sprague-Dawley rats compared to a control vector expressing GFP These data suggest that our virus-based in vitro screen can be employed to identify negative
in vivo modulators of TRPV1, providing insight into peripheral pain signaling and suggesting novel approaches for the control of primary hyperalgesia
Clinical Trial of a Granulocyte-Macrophage Colony-Stimulating Factor-Encoding, Second-Generation Oncolytic Herpes Virus (Talimogene Laherparepvec, T-VEC) in Patients with
Unresectable Metastatic Melanoma
John Nemunaitis,1,2,3,4 Cynthia Bedell,1 Beena O Pappen,4 Staci Horvath,1 Neil Senzer.1,4
1 Mary Crowley Cancer Research Centers, Dallas; 2 Medical City HCA, Dallas; 3 Texas Oncology, P.A., Dallas; 4 Gradalis, Inc, Dallas.
We previously conducted a multicenter phase II trial to assess the effi cacy of T-VEC in advanced stage IIIc and IV melanoma patients (Senzer, Nemunaitis et al JCO 2009) Treatment involved intratumoral (IT) injection of up to 4 mL of 106 pfu/mL of product followed 3 weeks later by up to 4 mL of 108 pfu/mL every 2 weeks for up to 24 treatments Clinical activity assessed by RECIST tumor response, survival and safety were monitored Fifty patients (stages
Trang 2Molecular Therapy Volume 21, Supplement 1, May 2013
Copyright © The American Society of Gene & Cell Therapy S185
IIIc, n = 10; IVM1a, n = 16; IVM1b, n = 4; IVM1c, n = 20) received a
median of six injection sets; 74% of patients had received one or more
nonsurgical prior therapies for active disease, including dacarbazine/
temozolomide or interleukin-2 (IL-2) The overall initial response
rate by RECIST was 26% (complete response [CR], n = 8; partial
response [PR], n = 5) Remarkably, despite technology being limited
to local-regional IT injection, both local and distal, non-injected,
metastatic sites (including visceral) demonstrated response Overall
survival was previously reported as 58% at 1 year We now report ³
5 year follow up of a subset of all 15 patients managed at MCCRC
Four of these patients were previously identifi ed as having achieved
complete response involving both local regional and metastatic
disease sites including lung and liver Three of these patients remain
alive 2528, 2031, and 1833 days, and one survived 868 days before
disease related mortality No long term adverse events were identifi ed
in all 15 patients including the 3 surviving ³ 5 years Five year Kaplan
Meier survival from time of fi rst treatment of the 15 patients treated at
MCCRC is shown below These results support long term durability
of response without evidence of adverse toxicity
Adenovirus Suppress Tumor Growth in Hamsters
Brittany A Young,1 Karoly Toth,1 Jacqueline F Spencer,1 William
S M Wold.1
1 Molecular Microbiology & Immunology, Saint Louis University
School of Medicine, St Louis, MO.
The Syrian hamster model has been described and used by our
laboratory and others for evaluating anti-tumor effi cacy, toxicity
and biodistribution of oncolytic adenovirus serotype 5 (Ad5)-based
vectors Hamster tumors and tissues are semi-permissive for Ad5
replication, which allows us to use permissive immunocompetent
animals for our studies We have previously reported that the
Ad5-based vector (VRX-007) replicates and suppresses tumor growth
when it is injected directly into subcutaneous tumors formed by
injection of hamster HaK renal cancer cells We also reported that
VRX-007 replication is increased and tumor growth is suppressed
when hamsters are treated with using cyclophosphamide (CP)
Effects that can be attributed to CP (an alkylating agent) include
immunosuppression of the hamsters that results in increased
VRX-007 replication and oncolysis in tumors as well as a chemotherapeutic
effect of CP on the tumor cells We now report that short-term CP
treatment, given for only one week before VRX-007 injection into
tumors, had a similar effect on enhancing the ability of VRX-007
to retard tumor growth as did long-term continuous CP treatment
Further, hamsters treated with short-term CP therapy did not have
detectable VRX-007 in their tumors, in contrast to modest levels
of VRX-007 in tumors of animals treated with CP continuously These results imply that (1) long term immunosuppression by
CP is not required for the CP-mediated enhancement of tumor growth control by VRX-007, and (2) that the CP may be acting as a chemotherapeutic agent in combination with VRX-007 but without increasing VRX-007 replication in tumors To further address these issues, we used a luciferase-expressing VRX-007 vector to view virus location and replication over time in HaK tumors via the IVIS
in vivo imaging system We found that short-term CP treatment, dosed either one week before or one week after tumor infection, had
an additive effect on VRX-007 tumor growth control even though virus replication in tumors was absent after one week Therefore, in this immunocompetent situation, the anti-tumor effects of VRX-007 and CP, alone or in combination, are exerted early after treatment and
do not involve long-term virus replication We have added radiation therapy as a third treatment modality to vector and CP therapies We found that tumor-specifi c irradiation has an additive effect with
VRX-007 therapy and further augments the inhibition of tumor growth, in both CP-treated and non-CP-treated animals, even though radiation does not lead to increased viral replication in tumors when compared
to those treated with virus alone These results further suggest that long term viral replication is not the cause for tumor growth inhibition
in our model This project aims to better understand the mechanism
of action of VRX-007 and possibly enhancing its anti-tumor effi cacy
481 Analysis of Host Cell and Viral Transcriptomes during Infection by Wild-Type Species C Ad6 and Species D Ad26
Mallory A Turner,1,2 Sean E Hofherr,3 Michael A Barry.2
1 Virology and Gene Therapy Program, Mayo Graduate School, Rochester, MN; 2 Department of Medicine, Mayo Clinic, Rochester, MN; 3 Laboratory Medicine, Children’s National Medical Center, Washington, DC.
There are more than 57 human Ad serotypes, which fall into seven species with diverse cell and tissue tropisms: A, B, C, D,
E, F, and G To date, species C Ad5 has dominated the fi eld of Ad based therapeutics However, in recent years limitations to Ad5 have emerged, including pre-existing immunity, blood factor affi nity, and off-target effects To combat these issues, many research groups are exploring the utility of vectors based on other Ad species and serotypes To better understand the differences in Ad serotypes, we compared the transcription and replication programs of of species D Ad26 and species C Ad6 in permissive cells qPCR for viral DNA showed that Ad6 and 26 have similar kinetics of viral DNA synthesis beginning between 6 and 12 hours after cell binding TruSeq mRNA libraries were generated for samples at 6 and 12 hours post infection and 101bp paired-end mRNA-seq was conducted 99.8% of 150 million reads were used to analyze both viral and host mRNAs Viral transcript sequences at 6 hours showed induction of Ad ‘early’ gene transcripts (E1, E2, E3, E4) for both Ad6 and Ad26 However, viral transcriptional output varied between Ad6 and Ad26 at some sites Notably, Ad6 E1A transcripts at 6 hours were about 4-fold higher than E1A transcripts of Ad26 Likewise, E3 total mRNAs varied between the two viruses in regions encoding homologous E3 proteins; fold changes varied between two and four E1B mRNAs were not signifi cantly different between the two viruses Adenoviral transcripts
at 12 hours post infection showed substantial increase in late gene transcripts (L1, L2, L3, L4, and L5) compared to the 6 hour time point
as expected In some cases Ad6 mRNAs exceeded those for Ad26 (L1, L3, L4) Interestingly, several presumptive early genes were not activated during the early phase, but were instead induced with the late genes mRNAs of the E2b region coding for DNA polymerase (pol) and the preterminal protein (pTP) were increased over 300-fold at 12 hours compared to 6 hours for both viruses The increase