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

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Presentazione sul tema: "Adenovirus and Adenoviral vectors Wild-type adenovirus contains a single, 36-kb, double- stranded DNA genome flanked by inverted terminal repeats. There."— Transcript della presentazione:

1 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.

2 Human adenovirus

3 Capside icosaedrico privo di envelope

4 Componenti strutturali

5 Major structural protein: the capsid II (hexon) Facets of icosahedron Major structural component, forms facets of the capsid III (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

6 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

7 Genome size 36 kb, dsDNS Produce più di 50 proteine, ori sono presenti nelle ITR (inverted terminal repeats), TP innesca linizio della replicazione The Ad genome 4,7 kb, ssDNA

8 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.

9 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

10 Ciclo vitale e trascrizione dei gni virali Fase precoce (espressione dei geni precoci E) Duplicazione del genoma Fase tardiva (espressione dei geni tardivi, L) Componenti del capside E:E1A, E1B, E2, E3, E4 Ritardati: IX, IVa2 L: processati in 5 RNA L1-L5 condividono il carbossi terminale polII transcript Richesti per la replicazione del genoma virale

11 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 step 1. Interazione fiber-knob con il CAR receptor 2.Interazione a più bassa affinità tra il motivo RGD della penton-base e le integrine di superficie Infezione target cells

12 CAR (coxsackie and Ad receptor): a major Ad attachment molecule Although CAR has been shown to support Ad entry to cultured cell lines, in the airway epithelium of the host, CAR expression is restricted to tight junctions and the basolateral membrane. Therefore there has been some debate as to whether CAR is accessible to the virus. CAR-independent virus entry cell-surface molecules have also been proposed to function as attachment sites for certain species C Ads including VCAM-1 (vascular cellular adhesion molecule-1) and HS-GAGs (heparan sulfate glycosaminoglycans)

13 Phagocytosis Adenovirus di tipo C fagocitosi mediata da clathrin-coated vescicles Adenovirus di tipo B micropinocitosi Internalizzazione

14 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. pIV

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

16 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

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

18 Oncogenesi Risposta immune: risposta non specifica iniziale con produzione di citochine; segue risposta specifica T citotossica diretta control le cellule infettate che espongono lantigene virale; attivazione dei linfociti B e risposta umorale % dell popolazione infantile ha anticorpi anti Ad serotitpi 1, 2 e 5 Attività trasformante del prodotto E1 la risposta immune dellospite

19 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 E1AE1B 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.

20 promotercDNA vettori deleti di E1 (E1D) possono essere prodotti in cellule packging che esprimono E1 in trans (293) 4,7-4,9 kb cDNA

21 prestazioni dei vettori adenovirali di prima generazione Derivano prevalentemente dal serotipo 5 Sono difettivi nella replicazione (rimoziene del gene E1 che è richiesto per la replicazione) Analisi delle barriere immunologiche al trattamento con vettori adenovirali somministrazione di un vettore adeno/lacZ per via biliare in: Topi immunocompetenti gli animali trattati mostravano 80% degli epatociti pos a 2gg dal trattamento, lespressione 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,

22 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.

23 Inoltre La somministrazione ripetuta dei vettori adenovirali era inefficace per linsorgenza di una risposta umorale a seguito della prima somministrazione con produzione di anticorpi che neutralizzano il virus impedendo di fatto linfezione delle cellule bersaglio per migliorare il potenziale terapeutico è stata proposta la eliminazione del gene E3 che codifica per proteine che modulano la risposta immunitaria

24 promotercDNA {} 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

25 second generation Ad vectors 1996 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

26 second generation Ad vectors E2A encods for a ssDNA binding protein essential for DNA initiation the first approach to improve 1 st 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

27 second generation Ad vectors the second approach to improve 1 st 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

28 gutted adenovector 2000 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.

29 "Gutted" adenoviruses Eliminazione di tutti I geni che agiscono in trans lasciando solo le sequenze necessarie per lincapsidazione A challenge for the Ad vector system is that the vector preparations are contaminated with low levels of helper Ad virus

30 changing target specificity molecular adaptor modifying the fiber

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

32 Produzione stock virale su larga scala Titolazione per plaque test

33 Ad &AAV application

34

35 Ad vectors

36 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 the capacity 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

37 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

38 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)

39 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 lefficacia 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 lattivazione del sistema immunitario indotta da vaccini geneticamente modificati.

40 Oncolytic virotherapy

41 Cell lysis

42 Strategia IV: virus oncolitici Virus oncolitici infettano e si replicano selettivamente nelle cellule tumorali ma non nelle cellule normali l

43

44 Virus oncolitici e p53 p53 induce arresto della crescita cellulare e apoptosi in risposta al danno al DNA p53 induce apoptosi anche in risposta allinfezione virale, così da impedire la propagazione del virus ai tessuti circostanti la proteina virale E1B-55kD blocca lattività 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

45 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.

46 Ad oncolitici la cui replicazione è limitata a cellule con p53 e Rb mutate ITR E1AE1A promoterE1B Ad Deleti per E1B (target p53 ricontrolla) o Ad con mutazioni in E1A (target co- activator of p53- oppure Rb tropismo ristretto ai tumori Si replicano in maniera selettiva nelle cellule tumorali

47 tumor selectivity One 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.

48 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

49 Cancer gene therapy and CAR down regulation In molti tumori si osserva una down regolazione del recettore CAR rendendo le cellule resistenti allinfezione da adenovirus Pseudotyping con proteine delle fibre di Ad che non utilizzano CAR (Ad35-CD46 che è upregolato in molti tumori)

50 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

51 Selettivita' per i tumori (virus oncolitici) Virus selvatico: ampio tropismo ITR E1AE1A promoterE1B Promotore prostata-specifico: tropismo ristretto a cellule prostatiche ITR E1APSA promoterE1B

52 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.

53 Studi clinici di terapia dei tumori con adenovirus oncolitici (ONYX-015) Modificata da Aghi e Martuza, Oncogene 24:7802:7816, 2005

54 Studi clinici di terapia dei tumori con adenovirus oncolitici Modificata da Aghi e Martuza, Oncogene 24:7802:7816, 2005

55 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 Oct;9(5):422-7 Hall et al Unity and diversity of the human adenoviruses: exploiting alternative entry patheways for gene therapy. Biochem J : 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)


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