Adenovirus and Adenoviral vectors

Wild-type adenovirus contains a single, 36-kb, double- stranded DNA genome flanked by inverted terminal repeats.

There are over 50 serotypes, from which serotypes 2 and 5 have been most developed for use as gene-therapy vectors.

This virus infects the upper respiratory tract, producing symptoms similar to those associated with colds and influenza, but as far as is known, it does not normally cause more serious disorders.

Human adenovirus

Capside icosaedrico privo di envelope

Componenti strutturali

Major structural protein: the capsid

II (hexon) Facets of icosahedron Major structural component, forms facets of the capsidIII (penton base) Capsid vertices Contains an RGD motif which facilitates interaction with cellular integrins*IV (Fibre) Projecting from the penton base Mediates the initial attachment to host cells

IIIa Underside of penton base Stabilizes the vertices

Inner hexon cavity Protease cofactor, assembly, endosome disruption and nuclear import of hexon

Between hexons Stabilization of peripentonal hexon–hexon interactions

IX External faces of the capsid Stabilization of virion. Transcriptional activator

Ad Core proteins

V Core Links core to capsid, possibly aids nuclear localization

VII Core Targets viral genome to the nucleus and condenses DNA

Mu Core DNA condensation

TP 5-End of the genome Primes DNA replication

IVa2 Core DNA packaging

Ad protease Core Cleaves precursor proteins

Major structural protein: the core proteins

Genome size36 kb, dsDNS Produce più di 50 proteine, ori sono presenti nelle ITR (inverted terminal repeats), TP innesca l’inizio della replicazione

The Ad genome

4,7 kb, ssDNA

adeno infection

• After infection, the viral DNA escapes from the lysosome and is transported to the nucleus of the cell, where it persists as an episome; multiple genomes can coexist within the nucleus of an infected cell.

• The adenoviral genome has eight transcriptional units, expressed in temporal sequence as early (E), intermediate (I), and late (L) genes.

• There are four early genes (E1–E4), encoding proteins necessary for the replication of the viral genome.

• E1A is the first viral gene expressed, and its product trans- activates the other promoters of early genes.

Ad genome organization

The rightward reading strand encodes the E1A, E1B, IX, the major late proteins VA RNA and E3 units.

The leftward reading strand contains the E4, E2A, E2B and IVa2 genes

blue arrows highlight intermediate

green arrows : late transcription units

red arrow: VA-RNA I and II

Early

Intermediate

Late

Ciclo vitale e trascrizione dei gni virali

Fase precoce (espressione dei geni precoci E) Duplicazione del genomaFase tardiva (espressione dei geni tardivi, L) Componenti del capside

E:E1A, E1B, E2, E3, E4Ritardati: IX, IVa2L: processati in 5 RNA L1-L5 condividono il carbossi terminale

polII transcript

Richesti per la replicazione del genoma virale

Mature virion

Attachment

Recettore CAR

C-term Fiber knob

Interazione penton-base e integrina (proteina di membrana)

Fiber knob-CAR

Iinterazione virione-cellule ospite, un processo a due step1. Interazione fiber-knob con il CAR receptor2.Interazione a più bassa affinità tra il motivo RGD della penton-base e le integrine di superficie

Infezione

target cells

CAR (coxsackie and Ad receptor): a major Ad attachment molecule

Although CAR has been shown to support Ad entry to culturedcell lines, in the airway epithelium of the host, CAR expressionis restricted to tight junctions and the basolateral membrane.Therefore there has been some debate as to whether CAR isaccessible to the virus.

CAR-independent virus entry

cell-surface molecules have also been proposed tofunction as attachment sites for certain species C Ads includingVCAM-1 (vascular cellular adhesion molecule-1) and HS-GAGs(heparan sulfate glycosaminoglycans)

Phagocytosis

Adenovirus di tipo C fagocitosi mediata da “clathrin-coated vescicles”

Adenovirus di tipo B micropinocitosi

Internalizzazione

The entry pathway of species C Ads

Attachment of the fibre knob to the primary receptor CAR. Subsequent interaction of the penton base with αv integrins. This leads to clathrin-mediated endocytosis resulting in virus internalization within endosomes

•The virus begins to dissociate in the low pH environment of the endosome and releases the vertex proteins including pVI. •Protein pVI can disrupt the endosomal membrane, allowing the partially dismantled virus particle to escape from the endosome. •The partially disassembled virus is then transported along microtubules by dynein to the nuclear pore complex. At the nuclear pore, viral DNA is imported into the nucleus.

http://www.BiochemJ.org/bj/431/0321/bj4310321add.htm

pIV

Alternative virus entry: the uptake of species B Ads into epithelial cells by macropinocytosis

Release from phagosome

Il DNA si associa con la matrice nucleare per favorire la replicazione

Ad viral replication require viral proteins that primer DNA synthesis – Tp primes

the synthesis

Cell lysis

Ad is a lytic virus: mature Ad virions are released following cell lysis

Oncogenesi

Risposta immune: •risposta non specifica iniziale con produzione di citochine; •segue risposta specifica T citotossica diretta control le cellule infettate che espongono l’antigene virale; •attivazione dei linfociti B e risposta umorale.

40-60% dell popolazione infantile ha anticorpi anti Ad serotitpi 1, 2 e 5

Attività trasformante del prodotto E1

la risposta immune dell’ospite

12S

13S

22S

DNA

RNA

E1A 13S

E1A 12S

E1B 19 KDa (antiapoptotica)

E1B 55 KDa

4 Kbp

La regione E1: essenziale per la regolazione della trascrizione e per la replicazione virale

E1A E1B

First genetion Adeno-vector:E1 deleted recombiant adenoviral vector

Delezione totale di E1a e parziale di E1b

The first-generation vectors were constructed by deleting the E1 and E3 regions of the adenoviral genome.

promoter cDNA

vettori deleti di E1 (E1D) possono essere prodotti in cellule packging che esprimono E1 in trans (293)

4,7-4,9 kb cDNA

prestazioni dei vettori adenovirali di prima generazione

Derivano prevalentemente dal serotipo 5Sono difettivi nella replicazione (rimoziene del gene E1 che è richiesto per la replicazione)

Analisi delle barriere immunologiche al trattamento con vettori adenoviralisomministrazione di un vettore adeno/lacZ per via biliare in:•Topi immunocompetenti gli animali trattati mostravano 80% degli epatociti pos a 2gg dal trattamento, l’espressione del transgene non era più rilevabile a 20gg dal trattamento•Topi atimici (non-immunocompetenti) non mostravano riduzione di espressione del transgene a 60gg dal trattamento

Questi risulatati suggeriscono che le cellule infettate dal vettore erano eliminate dalla risposta immune cellulo-mediate,

limitation of the first generation Ad vectors

The utility of these vectors, however, is limited by the continued synthesis of viral proteins by infected cells, despite the genetic deletions. These proteins render infected cells antigenic and thus liable to elimination by the immune system, a problem exacerbated by the subsequent discovery that the E3 domain of the virus encodes immunosuppressive proteins.

InoltreLa somministrazione ripetuta dei vettori adenovirali era inefficace per l’insorgenza di una risposta umorale a seguito della prima somministrazione con produzione di anticorpi che neutralizzano il virus impedendo di fatto l’infezione delle cellule bersaglio

per migliorare il potenziale terapeutico è stata proposta laeliminazione del gene E3 che codifica per proteine che modulano la risposta immunitaria

promoter cDNA

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vettori deleti di E1 ed E3 possono essere prodotti in cellule packging che esprimono E1 in trans (293)

4,7-4,9 kb cDNA 8,3 kb

Delezione di E3 aumenta la capacità

di clonaggio

second generation Ad vectors1996

Second-generation vectors have deletions in the E2 or E4 regions of the genome.

•These second-generation vectors are clearly improved with respect to immunogenicity and toxicity.•However, inactivation of proteins encoded by the E4 gene has been shown to impair seriously expression from heterologous promoters

second generation Ad vectorsE2A encods for a ssDNA binding protein essential for DNA initiation

the first approach to improve 1st Ad vector was to introduce a mutation into the E2A gene

•starting from a E1 vector•tsE2, temperature sensitive phenotype, lethal at 39 °C; expression of late proteins is reduced at the non permissive temp.

however, lack of persistence of Factor IX gene expression by this vector in animal model was observed

adenovirus genome

second generation Ad vectorsthe second approach to improve 1st Ad vector was to introduce a deletion of E4

E4 encodes for 7 ORFs that impact many events of the viral life cycle.

To allow vector production by E1 E4 vectors, the deleted functions need to be provided in trans.E4 was successfully expressed from an integrated copy within 293 cells (293-E4), the transgene was expressed by an heterologous promoter, so that to makes the expression of E4 independent from E1.Ad - E1 E4 vector

adenovirus genome

high level persistence expression of the transgene in vivo in animal models for at least

6 months

gutted adenovector2000

E’ possibile eliminare tutte le sequenze trans-agenti dal vettore?

Vettori adenovirali contenenti meno del 75% del genoma wt subivano riarrangiamenti.

Come superare questo inconveniente?

In the latest versions of adenoviral vectors, all viral coding sequences have been eliminated.

"Gutted" adenovirusesEliminazione di tutti I geni che agiscono in trans lasciando solo le sequenze necessarie per l’incapsidazione

A challenge for the Ad vector system is that the vector preparations are contaminated with low levels of helper Ad virus

changing target specificity

molecular adaptormodifying the fiber

Produzione del virus ricombinante in cellule packaging transfettate con il vettore virale contenente il gene di imteresse o con il vettore “guttled+ helper”

Produzione stock virale su larga scala

Titolazione per plaque test

Ad &AAVapplication

http://www.wiley.co.uk/genetherapy/clinical/

Ad vectors

Immune response•Ad vectors induced potent immune responses upon systemic application .

•Responses are directed against both the vector capsid and the low levels of Ad capsid proteins expressed from the vector

This limits transgene expression and reduces the capacity for vector re-administration

Such vectors induced potent immune responses upon systemic application [135,136].Responses are directed against both the vector capsid and the low levels of Ad capsid proteins expressed

from the vector. Induction of immune responses limits transgene expression and reduces thecapacity for vector re-administration. While the immunogenicity of these vectors may be reduced by the deletion of additional genes, the immunogenic nature of Ad vectors means that these viruses are better suited to applications for which prolonged transgene expression is not required. The use of Ad vectors in

vaccination to protect against a range of infectious pathogens has therefore proved to be a common strategy [137].

A significant proportion of gene therapy studies have also been designed to target cancers, where only short-term gene expression is required

Ad vectors application in

• Ad vectors in vaccination to protect against a range of infectious pathogens

• Ad vectors to target cancers, where only short-term gene expression is required

Ad against Cancer

Approach: inactivation of oncogenes and the addition of tumour suppressor genes or apoptosis-inducing genes.

Limitation: require the successful transduction of each cell within a tumour, which is currently not feasible.

Other Approaches:•Replication-deficient Ads with genes encoding secreted factors such as GM-

CSF (granulocyte macrophage colony-stimulating factor) and IL-12 (interleukin-12) to stimulate cytotoxic effects towards the tumour.

•Activation of apoptosis in non-transduced cells by soluble TRAIL (tumour-necrosis-factor-related apoptosisinducin ligand) encoded by an Ad5 vector

(bystander effect)

Effetto astante (bystander)

<< <<

• la possibilità di inserire geni terapeutici in masse tumorali è attualmente ristretta a trattamenti localizzati

• in questo ambito non tutte le cellule tumorali vengono trasdotte

E’ necessario mettere a punto sistemi che estendano l’efficacia del trattamento alle cellule vicine non trasdotte (effetto bystander)

Sono esempi di effetti bystander la diffusione di farmaci citotossici provenienti da cellule tumorali trasdotte con geni di suicidio e l’attivazione del sistema immunitario

indotta da vaccini geneticamente modificati.

Oncolytic virotherapy

Cell lysis

Strategia IV: virus oncolitici

Virus oncolitici infettano e si replicano selettivamente nelle cellule tumorali ma non nelle cellule normali

l

Virus oncolitici e p53

• p53 induce arresto della crescita cellulare e apoptosi in risposta al danno al DNA

• p53 induce apoptosi anche in risposta all’infezione virale, così da impedire la

propagazione del virus ai tessuti circostanti

• la proteina virale E1B-55kD blocca l’attività di p53, consentendo al virus di replicarsi

• Virus difettivi per E1B non riescono quindi a replicarsi nelle cellule normali perché è

attiva p53, ma riescono a replicarsi selettivamente nelle cellule tumorali con p53

inattivata

Cocchia 2002

The life cycle of Ad or herpes simplex virus (HSV)-1 can be divided into several stages.

During the infection stage, viral surface proteins, such as the adenovirus fibre or HSV glycoprotein D, mediate attachment to cellular receptors, such as coxsackie and adenovirus receptor (CAR) or HSV entry mediator C (HVEC), also known as

nectin 1. Once inside the cell, viruses express several gene products that target cellular

proteins and modulate various cellular processes, such as preventing apoptosis or inducing cell-cycle entry.

These promote viral replication and production of viral proteins that eventually lead to cell lysis and release of viral progeny. Each step is mediated by a diverse group of proteins. Examples of viral proteins and their cellular targets are provided. PKR,

double-stranded, RNA-dependent protein kinase; RR, ribonucleotide reductase.

Ad oncolitici la cui replicazione è limitata a cellule con p53 e Rb mutate

ITR

E1AE1A promoter E1B

Ad Deleti per E1B (target p53 ricontrolla) o

Ad con mutazioni in E1A (target co-activator of p53- oppure Rb

tropismo ristretto ai tumoriSi replicano in maniera selettiva nelle cellule tumorali

tumor selectivityOne approach to achieving tumour selective replication involves linking viral genes to promoters that are only

functional in tumour cells.

One tumour-specific promoter is derived from the gene that encodes alpha-fetoprotein (AFP). AFP is expressed in several tissues during development, but in adult tissues

its expression is limited to tumours of hepatic and intestinal origin.

In an adenoviral vector, this promoter can be used to regulate the expression of both E1A and E1B55kD. There is a 10,000-fold increase in the replication of this virus in AFP-expressing cells, compared with AFP-negative cells

Intravenous administration in mice causes regression of AFP-posi- tive tumours, such as hepatocellular

carcinomas.

I virus sono ingegnerizzati modificando le proteine virali di superficie che riconoscono recettori cellulari specifici, permettendo al virus di entrare in

maniera selettiva solo nelle cellule tumorali.

La selettività dei virus oncolitici per le cellule tumorali può essere incrementata agendo sulla specificità di infezione

Cancer gene therapy and CAR down regulation

In molti tumori si osserva una down regolazione del recettore CAR rendendo le cellule resistenti all’infezione da adenovirus

Pseudotyping con proteine delle fibre di Ad che non utilizzano CAR (Ad35-CD46 che è upregolato in molti tumori)

La selettività per la replicazione può essere ottenuta :

•utilizzando promotori specifici

•modificando i geni virali richiesti per una efficiente replicazione

La selettività dei virus oncolitici per le cellule tumorali può essere incrementata agendo sulla replicazione

Selettivita' per i tumori (virus oncolitici)

Virus selvatico: ampio tropismo

ITR

E1AE1A promoter E1B

Promotore prostata-specifico: tropismo ristretto a cellule prostatiche

ITR

E1APSA promoter E1B

The ONYX 015

Adenovirus depleted of E1B-55k protein and found that the replication phenotype was fully acquired in cells in which p53 was mutant, but not

in cells expressing a wild type p53. Based on these data, ONYX 015 was moved to the clinic and was

tested in several cancers.

Fase Tumore Adenovirus Chemiot. Risposte (anche parziali) Progressione tumorale

I Testa e collo ONYX-015 - 5/22 (23%) 9/22 (41%)

II Testa e collo ONYX-015 - 8/24 (33%) 7/24 (29%)

II Testa e collo ONYX-015 - 8/36 (22%) 15/36 (42%)

I Pancreas ONYX-015 - 6/22 (27%) 5/22 (23%)

I Glioblastoma multiforme

ONYX-015 - 0/24 23/24 (96%)

II Carcinoma orale ONYX-015 - N/S N/S

II Hepatobiliary ONYX-015 - 8/16 AFP (50%) 3/16 (19%)

I Ovarian ONYX-015 - 1/16 CA125 14/16 (88%)

II Carcinoma epatocellulare

ONYX-015 - 1/5 AFP 4/5 (80%)

II Cancro del colon metastatico

ONYX-015 - 4/18 CEA 11/18 (61%)

II Testa e collo ONYX-015 + 19/37 (53%) 5/30 at 6 months (17%)

Studi clinici di terapia dei tumori con adenovirus oncolitici(ONYX-015)

Modificata da Aghi e Martuza, Oncogene 24:7802:7816, 2005

Fase Tumore Adenovirus Chemiot. Risposte (anche parziali) Progressione tumorale

I-II Pancreas ONYX-015 + Partial response in 2/21 (10%) 11/21 (52%)

I-II Sarcoma avanzato ONYX-015 + 1/6 1/6

I-II Fegato ONYX-015 + CEA in 4/7 phase II patients 1/7 phase II cases by CT

I Metastasi epatiche di carcinoma del colon

ONYX-015 + 1/11 (patient received high dose) 0/3 at high dose; N/S for low dose

II Metastasi epatiche di carcinoma del colon

ONYX-015 + 3/27 partial response (11%), 4/27 MR (15%) 11/27 (41%)

I Metastasi polmonari ONYX-015 + 1/10 (10%) 1/10 (10%)

I Metastatico ONYX-015 + 0/9 (0%) 5/9 (56%)

I Prostata CV706 (PSA-driven E1A, E3-deleted)

- PSA 30% in 13/20; 4/20 (20%) with partial response (PSA 50% for 4 weeks)

N/S

I Solido H101 (E1B-deleted) - N/S N/S

I Prostata Ad5-CD/TK - PSA 25% in 7/16; 3/16 with partial response (PSA 50% for 4 weeks)

N/S

I Prostata Ad5-CD/TK - 15/15 with PSA ; 4/6 (67%) negative for adenocarcinoma at 1 year

N/S

II Solido H101 (E1B-deleted) - 14/46 (30%) 8/46 (17%)

Studi clinici di terapia dei tumori con adenovirus oncolitici

Modificata da Aghi e Martuza, Oncogene 24:7802:7816, 2005

Biblio

In neretto la bibliografia da consultare

Jiang et al. Oncolytic adenovirus: preclinical and clinical studies in patients with human malignant gliomas Curr Gene Ther. 2009 Oct;9(5):422-7

Hall et al Unity and diversity of the human adenoviruses: exploiting alternative entry patheways for gene therapy. Biochem J 2010 431: 321-336.

Russel 2012 “Oncolytic virotherapy” Nature Biotechnology

DONNA J. PALMER and PHILIP NG “Helper-Dependent Adenoviral Vectors for Gene Therapy, HUMAN GENE THERAPY 16:1–16 (2005)