AdenovirusesHost defenses viral countermeasuresHost defenses, viral countermeasures,

gene therapy

Elizabeth Moran, Ph.D.Professor of Orthopaedics

My contact info: MoranEL@umdnj.edu Office: Cancer Center Room G-1200 Phone: 2-5854

November, 2008Viruses, Cells and Diseases

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Office: Cancer Center Room G-1200 Phone: 2-5854

Adenoviruses: ChiefAdenoviruses: Chieffeatures

1. DNA genome.g

36,000 base pairs of DNA: small enough to be engineered effectively in the lab, and large enough to tolerate

~

incorporation of an expression sequence for a large mammalian protein without exceeding the capacity of its capsid structure.

CMV > Adeno > HPVCMV > Adeno > HPV10X 10X

2. Productive infection in a wide range of tissue types. 2

Clinical History ofClinical History of Adenovirus

Isolated from outbreaks ofIsolated from outbreaks of respiratory infections among military recruits in the 1950s

Over 50 serotypes are now known, divided into seven groups: A – G.

3Common lab serotypes (Ad2, Ad5) are type B

Ad i diAdenovirus diseases

The disease spectrum can vary depending on serotype; most common are cold symptoms, similar to rhinovirus infection.

Most people are sero-positive for adenovirus antibodies indicating they’ve had an infection. Adenoviruses are

id d i i l ll

Adeno can also cause eye infections.

widespread in animals as well.

In general, the most serious disease associated with Adeno is infant diarrhea, which can be severe, even life-threatening;

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, , g;these cases usually involve the enteric serotypes Ad40 and Ad41.

Adenovirus diseases

Adenovirus rarely causes serious illness in healthy immuno-competent adults.

However, . . .

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Ad14 is currently emerging as a virulent serotype capable of i f t l i i th i h lth lcausing fatal pneumonia in otherwise healthy young people.

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Why is adenovirus so importantWhy is adenovirus so important to molecular biologists ?

It was soon identified as a tumor virus

It grows very easily in cell culture, so has become a model lab organismhas become a model lab organism.

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Virus Interactions with the Host Cell

Adeno was instrumental in elucidating important concepts in the areas of:Adeno was instrumental in elucidating important concepts in the areas of:

i. Transcriptional regulation ii. Gene splicing as a mechanism to increase variability of gene

expression from a single gene.iii. Multi-step pathways to carcinogenesis and the interplay of

oncogene products and tumor suppressors.iv Host defenses and viral countermeasuresiv. Host defenses and viral countermeasures

iv Host defenses and viral countermeasures8

iv. Host defenses and viral countermeasures

Adenovirus structureAdenovirus structure

9Ad 5 binds to its receptor CAR (coxsackievirus and adenovirus receptor) through its fiber knob.

Adenovirus structureAdenovirus structure

Cell surface

Penton b

Fiber

Adenovirusvector

base

CAR integrin

Genetic engineering of the fiber protein

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g g pis being used to improve viral delivery in gene therapy.

The adenovirus genomeDNA

The adenovirus genome is a linear length of aboutThe adenovirus genome is a linear length of about 36,000 base pairs

How much coding capacity is this ??

H thi h di it b thHow can this much coding capacity be the foundation for an autonomous life form ??

11What design principles can be used ??

Virus life cycle

1 Vi t th ll d t it DNA1. Virus enters the cell and uncoats its DNA.

2. Virus early genes are expressed. At least one gene must be able to get itself transcribed in theprevailing conditions of the cell. - -

- - so virus immediate-early promoters are frequently used

3. Viral DNA is replicated.

y p q yfor constitutive overexpression of exogenous genes inmammalian cells.

DNA replication requires the expression of large numbers of enzymes.

4. Capsid proteins are made and assembled around the DNA.

If the cell is not replicating, the virus has to activate the genes that encode these enzymes.

Immediate early gene Early genes Late genes

DNA Replication

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y g

Timeline: 6-10 hours 8-24 hours 24-72 hours

Each capsid requires over 1600 protein molecules.

Virus life cycle

Role of the Early genes:y gi.Control expression of key viral and cellular genes

ii Counteract the ability of the cell to mount an anti viral responseii.Counteract the ability of the cell to mount an anti-viral response long enough to form new infectious units.

13Approximately 100,000 new viruses from a productively infected human cell

The adenovirus genome

Ad

E1A E1B E3L1 L2 L3 L4 L5

VA RNAs

Adeno genome

E4E2AE2B

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The adenovirus genome: E1A

Ad

E1A

Adeno genome

The E1A gene encodes two major products15

The E1A gene encodes two major products

E1A: seizes control of transcriptionE1A: seizes control of transcription

Two splice forms – same reading frame

13S TBP

Two splice forms same reading frame.

CR1 CR2 CR3CR4

13S

The 13S-unique region binds the TATA-Box Binding protein and is a promiscuous transcriptional activator – its main target is the other viral transcription units

12S

transcriptional activator – its main target is the other viral transcription units.

CR1CR4

12SCR2

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Key host cell regulators oftranscription

pRBE2F

transcriptionfactors

CyclinsCDKs

DNA synthesis factors

300p300 c-myc

Tissue-specificgene

expression

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Some immune response genes, e.g. intferon, MHC genes

E1A: seizes control of transcriptionE1A: seizes control of transcription

Two splice forms – same reading frame

13S TBP

Two splice forms same reading frame.

CR1 CR2 CR3CR4

3S

12S p300CR1

CR4

12S pRB CR2p300

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The 12S form mainly targets cell cycle specific genes. Quiescent cells use the pRB protein to repress the promoters of cell cycle genes. E1A binds pRB to compromise its repression function.

But unscheduled DNA synthesis is sensed by the cell and triggers an

apoptotic response. . .

p53 activates apoptosis-related gene expression

The Bcl2 protein down-regulates the apoptotic response.

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p53 induces transcription of the p21 cell-cycle i hibit d f t ti h Binhibitor, and of pro-apoptotic genes such as Bax.

Cyclin

p53 p21Cyclin

dependent kinases(CDKs)

Pro-apoptoticgenes

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Bax-related proteins form a complex that creates pores in mitochondria releasing caspase-activating factorsmitochondria, releasing caspase activating factors.

Wh B i b d bDNA damage signal When Bax is bound by Bcl2, Bax cannot form a

functional complexp53

DNA damage signal

BaxBcl2

BaxBax BaxBax

Bcl2Bax

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APOPTOSIS

The adenovirus genome: E1B

Ad

E1B

Adeno genome

The E1B gene encodes two major products22

The E1B gene encodes two major products

E1B 55K and 19K proteins prevent the cell f ti t t ifrom resorting to apoptosis

E1B: two splice forms – two different reading frames.

55K

19K

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E1B 55KE1B 55K

p53Promoter of cell cycle inhibitor yor pro-apoptotic gene

p53

The 55K protein binds p53 and prevents it from activating cell cycle inhibitors or pro apoptotic gene expression

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pro-apoptotic gene expression.

E1B 19KE1B 19K19K produced in abundance, sequesters most BAX

B l2 bi d dBcl2

19K

19K 19K

19K

Bcl2 binds and inhibits Bax

19K mimics Bcl22

B l2B

Bax

Bcl2Bax

Bax 19KBaxBaxBax

Bcl2Bax

Bax19KBax

BaxBaxBax

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Now the DNA replication process is activated, and the cells cannot opt out by apoptosis - but p y p phow to get DNA replication to act on the viral genome and not the cell genome?

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Th d i E2The adenovirus genome: E2

AdAdeno genome

E2AE2B

The E2 gene encodes three major proteins27

The E2 gene encodes three major proteins

E2 proteins: direct DNA synthesisto the viral genometo the viral genome

E2 encodes three proteins:80K pre-terminal binding protein (pTP)

attaches to 5” ends of viral DNA

72K DNA binding protein (DBP)binds to single stranded DNA – peels open the DNA for replication

140K DNA l140K DNA polymerase

Adeno DNA replication is primed by pTP,

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Cellular DNA replication is discontinuous;requires RNA primers.

and uses its own DNA polymerase.

DBP coats the DNA to separate the strands.

E2 proteins: direct DNA synthesisto the viral genome

These three proteins help adeno capture all the work of the host cell’s DNA synthesis machinery.

Adenovirus can produce about 100,000 new copies of its DNA in an infected human cell

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copies of its DNA in an infected human cell

Now you’ve replicated the viral DNA and are making hundreds of transcripts from the copies g p p- - -but the cell has mechanisms to restrict stability and translation of excess RNA

The interferon response evolved to recognize double-p gstranded RNA,

which is often associated with viral infection

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Cellular measures to restrict viral RNA (i t f )RNA (interferon)

Interferon-induced RNA-dependent protein kinase (PKR) binds double-Interferon induced RNA dependent protein kinase (PKR) binds doublestranded RNA.

The kinase becomes activated by auto-phosphorylation ( ) when it binds as P

a dimer.

The activated kinase targets a translational elongation factor (eIF-2) for inactivation by phosphorylation, and thereby shuts down protein synthesis

PP

IF 2

Viral RNA

+PKR

PP= eIF-2

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ProteinSynthesis

The adenovirus genome:The adenovirus genome:

The viral-associated (VA) RNAs

Ad

VA RNAs

Adeno genome

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VA RNAs: competitively inhibit cellmechanisms to restrict RNA

Viral-associated RNA I and II are transcribed in great abundance.

And are too small to permit PKR to bind as a dimer, so PKR becomes sequestered in an inactive form. ~ 165 nucleotides

P

PKR becomes sequestered in an inactive form.

PP

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Viral transcript

VA RNAs

VA RNAs: competitively inhibit cellmechanisms to restrict RNA

Viral-associated RNA I and II are transcribed in great abundance.

And are too small to permit PKR to bind as a dimer, so PKR becomes sequestered in an inactive form.PKR becomes sequestered in an inactive form.

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Viral transcript

VA RNAsMathews and Shenk (1991) J. Virol. 65:5657-5662

VA RNAs: do they also target theVA RNAs: do they also target theRNA interference (RNAi) system??

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mRNA degradation by small interfering RNAs

dsRNA miRNA

RNA interference involves a dsRNA cutting enzyme called Dicer, which cuts dsRNA made by viruses or formed from regulatory RNA Dicery g ysequences called microRNAs (miRNAs). Dicer cuts them into small pieces.

Dicer

The small dsRNA sequences unwind into small single strand RNAs (siRNA), which then combine with specific proteins to form an RNA induced silencing complex (RISC) Risc

small ds RNA

silencing complex (RISC). Risc captures complementary native mRNA molecules and cuts them up into untranslatable pieces.

RISCRISC

siRNA

mRNA

mRNA degradation by small interfering RNAs

dsRNA miRNA

Artificial siRNA sequences can be used to silence almost any gene.

DicerThis has tremendous potential clinical importance for gene therapy.

Dicer

small dsRNA

Can the the VA RNAs overwhelm the DICER or RISC enzymes

and impede siRNA therapy ?

RISC

and impede siRNA therapy ?

RISC

siRNA

mRNA

Aside from responses from the individual host cell the organism can also mount a systemiccell, the organism can also mount a systemicimmune response.

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Cells sample newly manufactured proteins, cut them into p y ppeptides and present the peptides on the cell surface using proteins of the Major Histocompatibility Complex

Killer T cells can recognizeKiller T cells can recognize foreign peptides, can become activated to attack any cells presenting those

Infectedcell

y p gantigens. Viral

peptideantigen

MHC

An environment of lysing cells

T cell

An environment of lysing cells with associated release of interferons, inflammatory factors and cytokines, such as TNFα will

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y ,activate killer T cells.

Th d i E3The adenovirus genome: E3

E3

Ad

E3

Adeno genome

The E3 gene encodes at least five small protein products40

The E3 gene encodes at least five small protein products

The viral antigens and MHC proteins are joined together in the endoplasmic reticulum on the pathway to presentation on theendoplasmic reticulum on the pathway to presentation on the cell surface

Viral peptide antigen

MHC

antigen

Endoplasmic reticulum

Golgi complex

Viral peptides

Proteosome

Viral protein

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The E3 19K glycoprotein tethers the MHC proteins in the endoplasmic reticulum

E3 19KE3 19K

Endoplasmic reticulum

Golgi complex

Viral peptides

Proteosome

Viral protein

42So no viral antigens are presented on the surface

But even non-antigen-presenting cells can be subject to cytolysis mediated by cytokinessubject to cytolysis mediated by cytokines

such as TNFα emitted by natural killer cells

TNFR1

Caspase activation is triggered by the interaction of the TNF family of death ligands

FADD

Pro-Caspase 8

(TNFα, Fas ligand, TRAIL)….with their corresponding death receptors, such as TNFR1.

CASPASE 3

APOPTOSIS

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The receptor cytosolic domains recruit adapter proteins, such as FADD, which then bind andactivate pro-caspase-8, which activates caspase 3 and causes cell death.

E3 proteins 10.4K and 14.5K associate to form a t i t li ti d d d ti (RID) lreceptor internalization and degradation (RID) complex.

Th RID lThe RID complex removes death receptors from the cell surface and marks them for degradation.

RID10.4K

14.5K

g

FADD

44E3 also encodes a 14.7K protein that protects further against TNF-induced cytolysis by inhibiting release of cytokines.

E3 DEATH Protein

Adeno resists apoptosis – but ultimately encodes an 11 6K nuclearAdeno resists apoptosis – but ultimately encodes an 11.6K nuclear membrane glycoprotein, regulated so as to accumulate only very late in infection, that has severe cytotoxic effect on cells;

. . . called the DEATH protein.

45Approximately 100,000 new viruses from a productively infected human cell

Th d i E4The adenovirus genome: E4

AdAdeno genome

E4

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E4 proteinsE4 proteins

E4 expresses at least six open reading frames (ORFs) that are mostly not essential for viral growth in cell culture, though the gene is

i d f ffi i t i l th i i llrequired for efficient viral growth in primary cells.

E4_ORF6 associates with E1B_55K to block transport of cellular RNA out of the nucleus in favor of viral RNA.

Other E4 ORFs help in functions such as capturing cellular transcription factors for viral promoters, and targeting anti-viral host proteins for degradation

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proteins for degradation.

Adenovirus early protein functions

E1A Takes control of viral and cell-cycle-related transcription.

E1B A 55 kDa protein binds and subverts p53A 19 kDa protein mimics Bcl-2

E d t l t f t i h ith l i di h t

E2 Encodes viral-specific DNA replication proteins, including a DNA polymerase

E3 Encodes at least four proteins, each with a role in evading host immune responses

Also encodes the DEATH protein, which accumulates to high l l l t i i f ti th i d ll l i t l tlevels late in infection, then induces cell lysis to release mature virus.

E4 Encodes about six small proteins with various functions that fine-tune interactions with host cellfunctions that fine tune interactions with host cell proteins.

VA RNAs Competitively inhibit interferon-induced shut-down of protein synthesis in response to double stranded RNA

In spite of all the virus countermeasures the host immunecountermeasures, the host immune

system usually does win out in the end

Most of us carry serum antibodies to adenovirus as evidence of past infections.

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How does this affect the ability to use adeno for gene therapy?

Adenovirus gene therapyAdenovirus gene therapy

Can selectively replicating lytic viruses be used as anti-tumor agents ?

i.e as oncolytic agents

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Hypothetical phenotype of an E1B (-) AdenovirusAdenovirus

In p53 (+) cells:In p53 (-) cells: p ( )

p53p53

Apoptosis would precedeViral assembly would

p53

p p pviral assembly.Infection would abort.

Viral assembly would Successfully precede lysis.Infection would continue.

NEIGHBORING CELLS would LIVENEIGHBORING CELLS would DIE

Adenovirus as a cancer therapeutic: Onyx -015 (H101)

Blue stain indicatesviral replication in the tumor;not in surrounding tissue,

i.e.: tumor cells (p53-negtive) die; normal cells (p53-positive) live.

Interestingly, though, the p53 state of the tumors does not really affect the outcome - - the specificity determinant may actually be the virus receptor - or the ability of tumor cells to complement- - the specificity determinant may actually be the virus receptor - or the ability of tumor cells to complement an E1B 55kDa-dependent function in progression from viral transcripts to translation.

A nearly identical virus, H101, was approved for human cancer treatment in China in 2006

Another oncolytic Adenovirus is based on an E1A mutation that makes E1A unable to bind pRB.

Adenovirus with E1A

mutation that makes E1A unable to bind pRB.

Adenovirus with E1A …unable to bind pRB

Virus cannot grow in these Vigrow in these cells

Virus can grow in, and lyse these cells

Normal cell pRB represses cell replication function Tumor cell

pRB is non-functional

Adeno-mediated gene therapy

FiberNon-lytic viruses can be used to deliver any desired coding

Penton base

Fiber deliver any desired coding sequence that can fit into the genome, if the target tissue has the proper receptors

CAR integrin

the proper receptors.

Potential uses:Potential uses:encode an enzyme to correct an inborn error

of metabolism or developmentencode an shRNA to target an unwanted

ll l

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cellular gene.others ….

Adenovirus and gene therapy

An E1a-deleted virus will not express other viral proteins well –well

so a virus with this background can be used as a vector t d li h f i i h llto deliver a human gene for expression in a human cell without much effect from the virus –

this requires a propagation cell line that complements E1A: 293 cells

55What other coding units are dispensable ?

Where to fit in a new gene for adeno to express ?.

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May lab stocks of adenovirus lack E3 –how does this affect use as a gene therapy vector?

Adeno carrying the HSV thymidine kinase gene can be used to activate the cancergene can be used to activate the cancer

chemotherapy pro-drug gancyclovir

CMVpromoter HSV thymidine kinase gene Adenovirus

vector

Viral kinase Cellular kinases

Gancyclovir

Toxic nucleotide

analogGancyclovirpro-drug

57DNA replication and cell proliferation blocked

This adenovirus is in phase 3 trials under the drug name Cerepro

How do you deliver your new gene therapy virus ?

Penton base

Fiber InhaleEat Inject

CAR integrin

Injectintra-tumorintravenous

integrin

Tumor cells tend not to have so many CAR receptors on their surface

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Adeno faces very different challenges when injected IV rather than inhaled or eatenIV, rather than inhaled or eaten

In cell IntravenousIn cellculture

Intravenousinjection

In 1999, an 18-year old patient, Jesse Gelsinger, participated in a trial at UPenn’s Inst. of Gene Therapy aimed at treating inborn ornithine transcarbamylase deficiency. He received a high dose of an adenovirus vector, delivered systemically, and died of massive

f il tl ltorgan failure, apparently as a result of a severe immune reaction.

This tragedy delayed gene therapy trials for years, while greater consideration was given to safer

Heparinaseconsideration was given to safer viral delivery.

59HSPG = heparin sulfate proteoglycans LRP = lipoprotein related protein

Read for DiscussionRead for Discussion

Baker AH, McVey JH, Waddington SN, Di Paolo NC, Shhayakhmetov DM. (2007). The influence of blood on in vivo adenovirus bio-distribution and transduction. Mol. Ther 15:1410-1416

Bauzon M, Hermiston TW (2008). Exploiting diversity: genetic approaches to creating highly potent and efficacious oncolytic viruses. Curr Opin Mol Ther. 10:350-355.

Some interesting news articles:

Branca MA (2005) Gene therapy: cursed or inching towards credibility? Nature Biotech. 23: 519-521Nature Biotech. 23: 519 521

Garber K (2006) China approves world’s first oncolytic virus therapy for cancer treatment.J. Natl. Cancer Inst. 98: 298-300.

Osborne R (2008) Ark floats gene therapy’s boat for now

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Osborne R (2008) Ark floats gene therapy’s boat for now.Nature Biotech. 26:1057-1059