Cerullo_verona2010_AdenovirusGT

Adenoviral Gene Therapy:applications and limitations

Vincenzo Cerullo, PhDDep. Cancer Gene TherapyUniversity of Helsinki

Verona, June the 24th 2010

Content of the lecture• Adenovirus• Adenoviral vectors

– Historical note– First Generation and Helper Dependent vectors– Oncolytic viruses

• Applications– Gene Replacement– Cancer Gene Therapy

Objective: Overview on Adenovirus and how it can be used in the context of Gene Therapy.

Adenovirus Structure

• Isolated in 1953• 50 different human serotypes,

divided in 7 groups (A-F)

• Unenveloped

• Icosaedric capsid (~ 90 nm)

• Nucleoprotein core

• dsDNA (~ 36 Kb)

• 12 fibers with terminal knob

Adenovirus Genome• Early genes

– They encode for proteins primarily concerned with modulating cellular metabolism to make the cell more susceptible to virus replication

– E1 Gene initiates the “machine Adenovirus”

• Late genes– They encode for structural proteins (e.g. protein of the capsid)

Adenovirus Entry

Adenovirus as Vector for Gene Therapy

Graham et al. Virology 52:456-67, 1973 Graham et al. J.Gen. Virol. 36:59-72, 1977

ψWt Ad

36 kbA

FG Ad

ψ

ΔE1B

HD Ad

ψC

Permissive cellsInfection, replication

E1(-) cells

E1(+) cells

Infection

Infection, replication

E1(+), Cre(+) cells Infection, replicationHelper Ad

ψ

ΔE1

Rb(+) cells

Rb(-) Cancer cells

Infection

Infection, replication

Cradψ

DΔ24

Wild Type adenovirus

Helper Dependent vector

First Generation vector

Oncolytic adenovirus

Applications

• Gene Delivery

– Monogenic disease

– In vitro studies

• Cancer Gene Therapy

• Vaccine• Delivery of antigens

Why Adenoviral vectors are an efficient tool for gene delivery?

• Easy to produce in high titer and stable• High capacity (up to 36 kb)• Infect efficiently quiescent cells and

differentiated tissues• High affinity for the liver • Do not integrate in host genome • Low immunogenicity (adaptive only for

HD-Ad)

Interaction between Adenovirus serotype 5 hexon and FX mediates liver Gene transfer

Waddington et al., Cell 132, 397–409, February 8, 2008

• In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells

• Coagulation factor (F)X directly binds adenovirus leading to liver infection

• FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface

Stable and sustained expression from HD-Ad vectors

Belalcanzar et al., Circulation. 2003;107:2726-2732.

PBS

Vector

• First example of long term expression mediated by a viral vector

• LDLR-/- mice were maintained on high fat diet and treated with saline or HD-Ad-ApoI

• HD-Ad-AI treatment of LDLR-/- mice leads to long-term overexpression of apoA-I, retards atherosclerosis progression, and remodels the lesions to a more stable-appearing phenotype.

Lifelong elimination of hyperbilirubinemia in the Gunn rat with a single injection of helper-dependent

adenoviral vector

• Crigler–Najjar syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by a deficiency of uridine diphospho-glucuronosyl transferase 1A1

• Total plasma bilirubin levels were reduced from >5.0 mg/dl to << 1.4 mg/dl for >2 yr after a single i.v. administration of vector expressing the therapeutic transgene at a dose of 3X1012 viral particles per kg

Toietta et al., PNAS 112:5, 3930-3935, March 15, 2005

HD-Ad can accommodate expression cassette up to 36 Kb

• 30% of patients receiving FVIII therapy develop neutralizing AB to the protein

• The size of the FVIII Expression cassette is about 14 Kb

• HD-Ad can accommodate WT FVIII and different variant of the protein able to overcome the formation of neutralizing antibodies

Cerullo et al., Mol Ther. 2007 Dec;15(12):2080-7

Long-term therapeutic liver trasduction with lack of toxicity

• Therapeutic dose of HD-Ad results in vector dissemination and severe toxicity

• Delivery of the vector into surgically isolated liver overcomes its dissemination and toxicity

• This approach resulted into stable and sustained expression in non human primates

Brunetti-Pierri et al., Hum Gene Ther. 2006 Apr;17(4):391-404 and Mol Ther. 2007 Apr;15(4):732-40

Pseudotyping of HD-Ad

• HD-Ad vector’s capsid depends on the Helper proteins

• Genetic modifications to the helper Virus will translate in modification to HD-Ad

• This Approach can be used to psudotyping HD-Ad vector producing chimeras and/or capsid modified vectors for retargeting to different tissues

Vectorψ

E1(+), Cre(+) cells

ψ

ΔE1

Helper Virus

ψ

ΔE1

Fiber

Knob Domain

B

B

BB

B

B

B

B

Modified Fiber System

Low dose of alpha 10 integrin Coupled with HD-AdLacZ transduce Articular Chondrocytes in vivo

PBS Complex WT Vector

Intradiscal injection of Complex augment Anulus and Nucleus transduction

Bgal Assay

1

10

100

1000

10000

100000

1000000

10000000

Bgal

Exp

ress

ion

LUx1

06 /m

g Pr

otei

n

C C C PBS WT WT

A N N A N A N A N A NA

C: Complex (Vector-10 Integrin)PBS: PBS injected diskWT: Wild Type fiber vector A: AnulusN: Nucleus Pulposa

Summary

• High capacity

• Liver tropism

• Stable and sustained expression from the liver

• Lack of long-term associated immune response in different species including non human primates

toxi

city

Vector administration

imm

unity

innate

adaptive

time

Adapted from Dr. Mane Thesis Defense (2006)

Fatal systemic inflammatory response syndrome in a OTC patient following FG-Ad gene transfer

• 18-year-old male with partial ornithine transcarbmaylase (OTC) deficiency

• 6X1011 VP/kg (3.8x1013VP total) of FG-Ad-OTC

• 18 hours post administration the patient was noted to have altered mental status and jaundice

• Intravascular coagulation, and multiple organ system failure, leading to death 98 h following gene transfer

• High transaminase and pro-inflammatory cytokines were noted during the clnical course of patient OTC-019

Raper SE et al., Mol Genet Metab. 2003 Sep-Oct;80(1-2):148-58

Acute Toxicity After High-Dose Systemic Injection of Helper- Dependent Adenoviral Vectors into

Nonhuman Primates• 2 baboon were administered with 5.6X1012 and 1x1013

VP/kg (3.4x1013VP total)• HD-Ad is devoid of all viral genes and no late toxicity

were showed in mouse model• High dose of HD-Ad resulted in severe/lethal toxicity in

non human primates

Brunetti-Pierri et al., Hum Gene Ther. 2004 Jan;15(1):35-46

toxi

city


imm

unity

innate

adaptive

time


toxi

city


imm

unity

innate

adaptive

Use of HD Ad

time


Early toxicity

toxi

city


imm

unity

innateadaptive

Use of HD Ad

time

How does the immune system detect and react to adenoviral vectors?

TLRs family are engaged to trigger the innate response (toxicity) to adenovirus

• TLRs is a family of receptors plays a critical role in the early innate immune response to invading pathogens by sensing microorganisms.

• These evolutionarily conserved receptors, homologues of the Drosophila Toll gene, recognize highly conserved structural motifs only expressed by microbial pathogens, called pathogen-associated microbial patterns (PAMPs).

• Here we showed that TLR9 is responsible for activating the innate immune system after delivering of HD-Ad

Cerullo et al., Mol Ther. 2007 Feb;15(2):378-85.

TLR2 and MyD88 are responsible for triggering part of the innate and adaptive response to adenovirus

Cerullo, Suzuki et al., Hum Gene Ther 2010, 21(3): 325-336

• Stimulation of TLRs by PAMPs initiates signaling cascades that involves a number of proteins, such as MyD88, TRIF and IRAK.

• These signaling cascades lead to the activation of transcription factors, such as AP-1, NF-κB and IRFs inducing the secretion of pro-inflammatory cytokines and effector cytokines that direct the adaptive immune response

• Here we showed that TLR2 and MyD88 are involved to innate and adaptive response to HD-Ad

MyD88MyD88

TLR2

Adenovirus

NFkB Activation

IRFs Transcription

NFkB

TLR9 Gene Upregulation

MyD88

TLR2

Adenovirus

Cytokines Production

NFkB Activation

IRFs Transcription

NFkB


MyD88

TLR2

Adenovirus


NFkB Activation

IRFs Transcription

NFkB Activation

NFkB


MyD88

TLR2

Adenovirus


NFkB Activation

IRFs Transcription

NFkB Activation

Nod2

0100200300400500600700800900

IL6 in NOD2 vs C57

IL6 (

pg/m

l)

Nod2 B6

*

The Complement system is involved to trigger innate and adaptive response to Adenoviral vectors

• The complement system is known to play critical roles in pathogen identification, initiation of the immune response

• In this study, they assessed the roles of the alternative (Factor B), classical (C1q and C4) and common (C3) arms of the complement system after vector administration

• Cytokines and chemokines secretion is C3 mediated• Thrombocytopenia is dependent on Factor B and C3• Humoral response to Ad capsid and its proteins are

complement mediated

Appledorn DM et al., Gene Therapy (2008) 15, 1606–1617

BL/6 C3-KO FB-KO C4-KO C1q-KO BL/6 C3-KO FB-KO C4-KO C1q-KO

Summary

• Although adenoviral vectors ensure high selectivity for the liver and sustained expression from this organ

• High dose of adenoviral vectors may cause severe toxicity that can lead to lethal complication

• Several innate immune sensors (TLRs, NODR, complement) are responsible for triggering the “early toxicity” to adenoviruses

Applications

• Gene Delivery

– Monogenic disease

– In vitro studies

• Cancer Gene Therapy

• Vaccine• Delivery of antigens

Adenoviral Based Cancer Therapy

• Vectors mostly based on Ad5 Limited pathogenicity for humans Broad host cell range High replication efficiency Replication results in cell lysis and spread of the progeny

to neighboring cells Genome relatively easy to manipulate (inserts up to 10 kb) Ability to generate high titers

• Several clinical trials • H101 licensed in China in 2005

Oncolytic Adenoviral Vectors

• Redirect viral tropism Genetic Retargeting Transductional Retargeting

• Conditionally replicating adenoviruses (CRADs) Loss-of-function mutations in essential genes (E1A, E1B, VA) Promoter-inducible CRAds (using TSP)

• Arming Insertion of transgenes coding transgenes for cytokines,

prodrug-activating enzymes or cytotoxic proteins

St. James University

Clinical Trial around the world

Targeting of Tumor cells• Transductional targeting (fiber-pseudotyping)

– Ad5/3 Chimera (Krasnykh et al. J Virol 1996;70: 6839-6846)

– pK7 HI-loop (Wickham et al. J Virol 1997;71: 8221-8229)

– RGD modification (Mizuguchi et al. Gene Ther 2001;8: 730-735)

• Transcriptional targeting– CEA promoter to drive E1A gene (Brand et al Gene Ther

1998; 5: 1363-1371)

– COX-2 (Yamamoto et al. Mol Ther 2001; 3: 990-996)

• Biological targeting – p53 (DE155K – ONYX-015)

– Rb (D24 – CGCTs viruses)

DNA Damage

p53P

p53Mdm2

Apoptosis and cell cycle arrest

p53Pp53

P

p53P

p53P

p53

RbE2F

Rb

P

E2FE2F

E2F

S Phase

Mdm2

Mdm2

Mdm2

p53

p53

p53

p14ARF

Apoptosis and cell cycle arrestCell cycle in Normal cell

Mitogen

RbE2F p53

p53p53


E1A

E1B55K

E1B55K

E1B55K

E1A

Rb E1A

E2F

E2F

E2F

E2F

Wild Type Adenovirus in Normal cell

Rb

p53p53


1E AD24

E2F

DE1B55K

E1B55K

E1B55K

p53

p53 p53

1EAD

24

Rb E2F

Oncolytic Adenovirus in Normal cell

1E AD24

DE1B55K

Oncolytic Adenovirus in Cancer cell

p53p53

Apoptosis and cell cycle arrestE2F

p53

p53 p53

Rb E2FRb

S phase and replication

Summary

• Oncolytic Adenovirus is able to infect all permissive cells

• Oncolytic adenoviruses can replicate only in tumor cells (p53, Rb mutated pathway)

• Oncolytic adenoviruses DO NOT replicate in normal cells

An mutant adenovirus that replicates preferentially in p53 mutated cells

Bischoff et al., Science 18 October 1996: 373

• The human adenovirus E1B gene encodes a55-kilodalton protein that inactivates the cellular tumor suppressor protein p53

• Here it is shown that a mutant adenovirus that does not express this viral protein can replicate in and lyses p53-deficient human tumor but not cells with functional p53

ONYX-015, an E1B gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral

efficacy

• Human normal cells are highly resistant to ONYX15-mediated replication-dependent cytolysis

• A wide range of human cancer cell lines, with either normal or mutated p53 sequence, were shown to be sensitive to ONYX15

• Efficacy of the virus was assessed in nude mice bearing human tumors, survival analysis was performed

Heise et al., Nat Med. 1997 Jun;3(6):639-4

ONYX-015 in clinic (Phase I)

• Phase I trials (Ganly et al 2000, Mulvihill et al 2001) demonstrated safety at all used doses (manufacturing capabilities were the only limitation)

• 14% PR (3 of 22 patients) and 45% SD (10 of 22 patients)

ONYX-015 in clinic (Phase II)• Phase II trials conducted on patients with

recurrent HNSCC.• Two different doses regimen:

– Standard (i.t. daily for five days)– Hyperfractionated (twice a day for two weeks)

• 14% objective response (including 3 out of 40 patients with CR)

• 41% (12 patients) showed SD• No differences between the regimens• Responses significantly correlated with p53

status

D24 deletion in E1A gene of adenovirus results in potent and selective anti-tumor efficacy

• They identified a E1A (D24 deleted) mutant that replicates and lyses a wide range of tumor cells with abnormalities in cell-cycle checkpoints

• The mutant demonstrated reduced S-Phase induction and replication in non-proliferating normal cells

• Its potency in vivo was superior compared to other gene-deleted adenovirus and even superior to wild-type adenovirus

Heise C et al., Nature Medicine 6, 1134 - 1139 (2000)

PBSD24 VirusONYX VirusWild-Type

Armed Oncolytic Adenoviruses

• Transgene expression from E3 region is feasible• Expression of the E3 genes can be retained• In insertion site choice can effect both the transgene

expression levels and life cycle• The expression levels is dependent on viral DNA

replication

Hawkins LK et al., Gene Therapy (2001) 8, 1123–1131

Ad5D24-GMCSF

Cerullo et al., Cancer Res. 2010 Jun 1;70(11):4297-309

Ad5D24-GMCSF stimulates tumor-specific immune response in Syrian hamsters


Tumor response and survival


Non injected tumor in mesothelioma patient M50 (SD)

Complete Response in Ovarian cancer Patient (O14).

Complete Response in breast cancer Patient (R8).

Rapid response in ovarian cancer patient (O9)

Ad5D24-GMCSF stimulates tumor-specific immune response in Cancer Patients


Conclusions

• Oncolytic Adenoviruses preferentially replicate in cancer cells

• Cell lyses occurs as consequence• Cell lyses releases tumor antigens that may be

processed and loaded onto MHC-I• Hundreds of clinical trials to date have

demonstrated safety in human patients • Increasing the efficacy is still needed (new agents

and choosing of better cohorts of patients)

Prof. Francesco SalvatoreProf. Lucio PastoreDr. Barbara Lombardo Prof. Brendan Lee

Prof. Phil NgProf. Nicola Brunetti-PierriDr. Michael SeilerDr. Masataka Suzuki

Prof. Akseli HemminkiAll the postdocsAll the students (Iulia Diaconu, Matteo Ugolini)All the technicians (Sirkka Liisa-Holm)

Thank you

Prof. Vladia Monsurro’

Collaborations

Production of First Generation vectors NucleusCytoplasm

E1

Production of HD-Ad vectors NucleusCytoplasm

E1 + CRE

Vector

ψ

E1(+), Cre(+) cells

ψ

ΔE1

Helper Virus

ψ

ΔE1

Production of Oncolytic VirusesNucleusCytoplasm

Rb/p53

6x1011VP/Kg => 3.8x1013VP total

1.1x1013VP/Kg => 3.4x1013VP total

Education

Cerullo_verona2010_AdenovirusGT