Adenovirus vectors

1
Adenovirus vectors

• Gene Therapy-2008

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Virology 1

• Infectivity
• How do you measure it?
• Viral particles
• How do you count them?
• Quality control
• Why is it important?
• Host range
• What determines it?

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Infectivity

• Measure by infecting dilutions of the virus stock
  on permissive (e.g. 293) cells.
• Plaque assay (1-2 weeks)
• Foci of infection
• Rapid titer (2 days)
• Measure by immunostaining viral antigens
• Endpoint dilution
• TCID50
• FACS after immunostaining viral antigens

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Infectivity (continued)

• Infectivity is variable and dependent upon
• Passage number and health of cell substrate
• Time of virus adsorption
• Volume of virus adsorption

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Poisson distribution

• The equation for the Poisson distribution is
• Px(mx.e-m) / x!
• Where Px the probability of a cell being "hit"
  by x viruses
• m   mean hits per cell multiplicity of
  infection (moi).
• e     natural logarithm.

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Poisson distribution (continued)

• Therefore at an MOI of 1, P(0)0.37
• This means that at 1 PFU/cell approx. 63 of the
  cells are infected.
• Therefore one typically infects at an MOI of 10
  to infect all the cells.

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Virus purification

• Ultracentrifugation
• Rate zonal
• Dependent upon sedimentation velocity (size and
  shape) of virus particle. Underutilized.
• Isopycnic
• Dependent on equilibrium density of virus
  particle.
• CsCl typical
• Buoyant density of adenovirus is 1.35 g/ml
• Determine by refractometry

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Particle number

• Measured by A260
• Quantitative PCR
• Is invariant and therefore a good number to use
  when infecting cells
• Particle/PFU ratio typically 10-100
• Doesnt necessarily infect amount of dead virus
  in preparation

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Quality Control

• Replication competent adenovirus
• Sterility
• Titer
• Seed lot system
• Virus
• Cells

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Adenovirus structure
Adenovirus ds DNA approx. 36 kd with a terminal
protein attached
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Adenovirus transciption
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Function of transcripts

• E1 Transactivation of early genes
• E2 Adenovirus DNA replication
• E3 Prevention/modulation of host immune
  response
• E4 Prevention/modulation of CTL response

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Adenovirus receptors

• Fiber
• Spike of the virus
• Binds coxsachie-adenovirus receptor (CAR) in
  sub-group C
• Binds CD46 in subgroup B
• Penton base
• Co-receptor-binds RGD (integrin) for
  internalization of virion

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Adenovirus Receptors (continued)

• Receptors are not fully understood
• In vivo distribution doesnt match receptor
  distribution
• Other serotypes (e.g.type 37, ocular) may have
  other receptors (e.g. sialic acid)
• Certain serotypes bind clotting factors
• Small molecules may be involved in adenovirus
  host range (adamantine).

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Adenovirus Host Range

• Adenovirus infects humans
• No other good animal model
• Chimpanzee
• Monkeys are non-permissive
• Mouse model is poor because most murine cells
  lack viral receptors
• Host range can be enhanced by using lipids
  containing polyamines (see Davis, Human Gene
  Therapy, 2005)

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Subgroups, serotypes, and disease
Adenovirus serotypes and clinical disease
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Adenovirus in gene therapy
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Initial safety and immunogenicity Vaccine
(1992) studies of an oral recombinant adenohepatit
is B vaccine Carol 0. Tacketi, Genevieve
Losonsky, Michael D. Lubeck?, Alan R.
Davis?, Satoshi Mizutani, Gary Horwith, Paul
Hung, Robert Edelman and Myron M.
Levine Orally administered adenovirus may be a
useful vaccine carrier of cloned antigens of
other pathogens. A recombinant adenohepatitis
vaccine Wy-Ad7HZ6-I, which expressed hepatitis B
surface antigen and contained a large deletion in
early region 3 (E3), was constructed and studied
in humans. Volunteers received Wy-Ad7HZ6-I (n
3), adenovirus type 7 vaccine (n 3) or placebo
(n 3). Recipients of Wy-Ad7HZ6-1 shed less
vaccine virus in the stool for a shorter period
and had a lower titre of anti-adenovirus type 7
antibodies than recipients of the adenovirus 7
vaccine. None of the three Wy-Ad7HZ6-I vaccinees
developed antibody to hepatitis B surface antigen
after this one dose primary immunization regimen.
The E3 region may be required for optimal
enteric growth of adenovirus-vectored
vaccines. Center for Vaccine Development,
Department of Medicine, University of Maryland
School of Medicine, Baltimore, MD 21201, USA.
Wyeth-Ayerst Research, Philadelphia, PA 19101,
USA. To whom correspondence should be addressed.
(Received 10 December 1991 revised 7
February 1992 accepted 10 February 1992)
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Adenovirus-HIV vaccine
Title Recombinant adenovirus vaccines Document
Type and Number United States Patent 20040170647
Kind Code A1
AR Davis, MD Lubeck, RJ Natuk, PK Chanda,
Abstract This invention provides a method of
protecting a primate from an infectious organism
by stimulating the production of antibodies or
cell mediated immunity to the infectious organism
which comprises administering to said primate
intranasally, intramuscularly, or subcutaneously,
live recombinant adenoviruses in which the virion
structural protein is unchanged from that in the
native adenovirus from which the recombinant
adenovirus is produced, and which contain the
gene coding for the antigen corresponding to said
antibodies or inducing said cell mediated
immunity. Preferably, the infectious organism is
HIV and the primate is a human.
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Clinical Testing
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Current Gene Therapy Clinical Studies Using
Adenovirus Vectors

• 1. RecruitingAdenovirus and Fungal Load in
  Pediatric Stem Cell Transplant PatientsConditions
  Adenovirus Other Mycoses
• 2.RecruitingVirus-Specific Cytotoxic
  T-Lymphocytes (CTLs) for Adenovirus Infection
  Following an Allogeneic Stem Cell
  TransplantCondition Adenoviridae Infections
• 3.RecruitingSafety and Immunogenicity of
  Recombinant DNA and Adenovirus Expressing L523S
  Protein in Early Stage Non-Small Cell Lung
  CancerCondition Non-Small Cell Lung Cancer
• 4.RecruitingSafety of and Immune Response to a
  DNA HIV Vaccine Followed by an Adenoviral Vaccine
  Boost Given 3 Different Ways to HIV Uninfected
  AdultsCondition HIV Infections
• 5.RecruitingAdenovirus Vaccine for
  MalariaCondition Malaria
• 6.RecruitingSafety and Efficacy of a Three-Dose
  Regimen of an Adenoviral HIV Vaccine (MRKAd5
  HIV-1 Gag/Pol/Nef) in HIV Uninfected South
  African AdultsCondition HIV Infections
• 7.RecruitingAdministration of Autologous DCs
  Infected with an Adenovirus Expressing
  Her-2Conditions Inclusion criteria Patients
  with metastatic breast cancer who are HER2/neu
  positive (3 by immunohistochemistry or FISH
  positive) and either, ...
• 8.RecruitingAdenovirus Encoding Rat HER-2 in
  Patients With Metastatic Breast Cancer
  (AdHER2.1)Conditions Metastatic Breast Cancer
  Recurrent Breast Cancer
• 9. .RecruitingWild Type p53 Adenovirus for Oral
  PremalignanciesConditions Mouth Cancer
  Dysplasia/Carcinoma in Situ (CIS) of the Oral
  Cavity Dysplasia/Carcinoma in Situ (CIS) of the
  Oral Pharynx
• 10.RecruitingStudy to Evaluate the Safety and
  Efficacy of Adeno-IFN Gamma in Cutaneous B-Cell
  LymphomaCondition Lymphoma, B-Cell
• 11.RecruitingNMRC-M3V-Ad-PfCA Vaccine - Clinical
  Trial 1Condition Plasmodium Falciparum
• 12.RecruitingChemotherapy Followed By Vaccine
  Therapy in Treating Patients With Extensive-Stage
  Small Cell Lung CancerCondition Lung Cancer
• 13.RecruitingVaccine Therapy With Either
  Neoadjuvant or Adjuvant Chemotherapy and Adjuvant
  Radiation Therapy in Treating Women With
  p53-Overexpressing Stage II or Stage III Breast
  CancerCondition Breast Cancer
• 14.Not yet recruitingSafety of and Immune
  Response to an HIV DNA Plasmid Vaccine Followed
  by HIV Adenoviral Vector Vaccine in Healthy
  African AdultsCondition HIV Infections

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15.RecruitingSafety of and Immune Response to a
DNA HIV Vaccine Followed By an Adenoviral Vector
HIV Vaccine in Healthy AdultsCondition HIV
Infections 16.RecruitingInterleukin-12 Gene in
Treating Patients With Liver Metastases Secondary
to Colorectal CancerConditions Colorectal
Cancer Metastatic Cancer 17.RecruitingBiological
Therapy in Treating Women With Breast Cancer That
Has Spread to the LiverConditions Breast
Cancer Metastatic Cancer 18.RecruitingPhase I
Study of Vaccination Schedule of Experimental HIV
VaccinesCondition HIV Infections 19.RecruitingPh
ase I Study Combining Suicide Gene Therapy With
Neoadjuvant Chemoradiotherapy in the Treatment of
Potentially Resectable Pancreatic
AdenocarcinomaCondition Pancreatic
Cancer 20.RecruitingGene Therapy for Prostate
Cancer That Returns After Radiation
TherapyConditions Prostatic Neoplasms Neoplasm
Recurrence, Local 21.Not yet recruitingSafety
Study on the Transfer of the CD40 Ligand Gene
(AdcuCD40L) to Patients With Esophageal
CarcinomaCondition Esophageal
Neoplasms 22.RecruitingNeoadjuvant Capecitabine
and Radiation Therapy With or Without TNFerade
Followed By Surgical Resection in Treating
Patients With Stage II, Stage III, or Locally
Recurrent Rectal CancerCondition Colorectal
Cancer 23.RecruitingEffect of AdhAQP1 on Salivary
Flow in Patients Treated With Radiation for Head
and Neck CancerCondition Parotid Salivary
Dysfunction 24.RecruitingGene Therapy for Pleural
MalignanciesConditions Pleural Mesothelioma
Metastatic Pleural Effusions 25.RecruitingSafety
and Immune Response to a Prime-Boost Vaccination
Schedule in HIV-Infected PatientsCondition
HIV 26.RecruitingA Clinical Trial to Evaluate the
Safety, Efficacy, and Immunogenicity of
DR-5001Condition Respiratory Tract
Diseases 27.RecruitingVector Delivery of the
IL-12 Gene in Men With Prostate
CancerConditions Prostatic Neoplasms Prostate
Cancer 28.RecruitingVaccine Trial for Clear Cell
Sarcoma, Pediatric Renal Cell Carcinoma, Alveolar
Soft Part Sarcoma and Children With Stage IV
MelanomaConditions Sarcoma, Clear Cell
Sarcoma, Alveolar Soft Part Renal Cell
Carcinoma Secrete Granulocyte-Macrophage
Colony-Stimulating Factor (GM-CSF
Melanoma 29.RecruitingVaccination With Autologous
Breast Cancer Cells Engineered to ) in Metastatic
Breast Cancer PatientsCondition Breast
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Cancer 30.RecruitingHer-2/Neu in Patients With
Metastatic Breast Cancer (AdHERe)Conditions
Metastatic Breast Cancer Locally Recurrent
Breast Cancer 31.RecruitingStudy of Treatment and
Metabolism in Patients With Urea Cycle
DisordersCondition Amino Acid Metabolism,
Inborn Errors 32.RecruitingSafety of and Immune
Response to an HIV-1 Vaccine (VRC-HIVDNA016-00-VP)
and a Vaccine Booster (VRC-HIVADV014-00-VP) in
HIV Uninfected East African AdultsCondition HIV
Infections 33.RecruitingDose-Escalation Study of
CG0070 for Bladder Cancer After BCG (Bacillus
Calmette-Guerin) FailureConditions Carcinoma,
Transitional Cell Bladder Neoplasms 34.Recruiting
Safety and Efficacy Study of INGN 241 Gene
Therapy in Patients With In Transit
MelanomaConditions Malignant Melanoma Neoplasm
Metastasis 35.RecruitingExperimental Vaccine for
Prevention of Ebola Virus InfectionConditions
Ebola Hemorrhagic Fever Ebola Virus Disease
Ebola Virus Vaccines Envelope Glycoprotein,
Ebola Virus Filovirus 36.RecruitingGene Therapy
in Preventing Cancer in Patients With
Premalignant Carcinoma of the Oral Cavity or
PharynxCondition Head and Neck
Cancer 37.RecruitingStudy to Compare the Overall
Survival of Patients Receiving INGN 201 (Study
Drug) With Patients Receiving MethotrexateConditi
on Carcinoma, Squamous Cell 38.RecruitingEffectiv
eness and Safety of INGN 201 in Combination With
Chemotherapy Versus Chemotherapy AloneCondition
Carcinoma, Squamous Cell 39.RecruitingPhase I -
Pre-Radical Prostatectomy RTVP-1 Gene Therapy for
Prostate CancerCondition Prostatic
Neoplasms 40.RecruitingA Study of TNFerade
Biologic With 5-FU and Radiation Therapy for
First-Line Treatment of Unresectable Locally
Advanced Pancreatic CancerCondition Pancreatic
Cancer
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Safety and Efficacy of a Three-Dose Regimen of an
Adenoviral HIV Vaccine (MRKAd5 HIV-1 Gag/Pol/Nef)
in HIV Uninfected South African Adults This study
is currently recruiting patients.Verified by
National Institute of Allergy and Infectious
Diseases (NIAID) December 2006

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Safety and Efficacy of a Three-Dose Regimen of
an Adenoviral HIV Vaccine (MRKAd5 HIV-1
Gag/Pol/Nef) in HIV Uninfected South African
AdultsThis study has been suspended. ( Based
on an interim data review, the DSMB concluded
that the vaccine cannot be shown in this trial
to prevent HIV infection or affect the course of
the disease. )
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Bone formation in a wild type mouse
2500 vp/cell Ad5BMP2
C57BL\6
Genejammer
Genejammer
C57Bl\6 derived cell line.

• Bone formation only occurs if the cells are
  effectively transduced with the virus. This step
  is critical.

- Genejammer
Fouletier-Dilling et al Human Gene Therapy
16(11) 1287-1298.
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Efficient and Rapid Osteoinduction in an Immune
Competent Host

• No differences in host cell survival
• No differences in the volume of bone formed
• No differences in stages of bone formation
• No differences in the timing of bone formation

Fouletier-Dilling et al Human Gene Therapy 18 .
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Stages of Rapid Endochondral Bone Formation
VWF
Day 1 Appearance of brown fat
Day 3-4 Angiogenesis and stem cell extravasation
Collagen type II
Day 7 Mineralized bone
Day 5-6 Appearance of cartilage
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Hydrogel encapsulation of Ad5BMP2 transduced cells
Osteoclast specific degradation sites
PEG-DA polymer chain

cells
PEG-DA photoinitiator solution
cell suspension
aliquots added to cell culture dish
exposure to white light, 1-1.5 min
photopolymerized PEG-DA chains
cells encapsulated within PEG-DA hydrogel
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Development of target delivery of the AdBMP2
transduced cells
Ad5F35BMP2
Encapsulation
Transduce
30µL hydrogel disks in a sheet structure
150µL hydrogel disk
Implant the disks near the critical size defect
Comparison of BMP2 secretion from the hydrogel
encapsulated and unencapsulated cells
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Hydrogel encapsulation of Ad5BMP2 transduced cells
Osteoclast specific degradation sites
PEG-DA polymer chain

cells
PEG-DA photoinitiator solution
cell suspension
aliquots added to cell culture dish
exposure to white light, 1-1.5 min
photopolymerized PEG-DA chains
cells encapsulated within PEG-DA hydrogel
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Other

• Slides of this lecture are available on our
  website
• http//vector.bcm.tmc.edu/
• References (will be fair game on exam)
• Biology of adenovirus and its use as a vector for
  gene therapy. Hum Gene Ther. 2004
  Nov15(11)1022-33. McConnell MJ, Imperiale MJ
• Adenovirus from foe to friend. Rev Med Virol.
  2006 May-Jun16(3)167-86 Goncalves MA, de Vries
  AA.