Adenoviruses and viral gene therapy vectors

Question 1

Structure of adenovirus

Answer 1

Naked
icosahedral
linear, double-stranded DNA

Question 2

What kinds of infections do adenoviruses cause?

Answer 2

persistent infections in the tonsils, adenoids, and other lymphoid tissues of humans

pharyngitis, tonsillitis, and nasopharyngitis

Question 3

Are adenoviruses pretty hardy or sensitive?

Answer 3

Pretty hardy
Relatively stable in homogenates of infected cells.
Stable to heat
No lipid so are resistant to lipid solvents, including bile salts
For this reason, adenoviruses survive the conditions prevailing in the lower gastrointestinal tract and can often be isolated from the feces as well as from the throat.

Question 4

Adenovirus replication

Answer 4

Model for gene regulation.

The virus attaches to the cell via the fiber protein and is internalized via receptor-mediated endocytosis in a manner dependent on interaction of the penton base with specific integrins.

Early gene products promote the cell’s transition into S-phase and activate viral genes for viral DNA replication.

The mechanism of viral DNA replication involves covalent attachment of a virally encoded protein, terminal protein, to the first base of the 5’ end of each DNA strand to serve as a primer for the virally encoded DNA polymerase. Thus, adenovirus DNA replication offers a tempting target for antiviral drug therapy.

Question 5

Immunological characteristics of adenovirus

Answer 5

Hexons possess a type-specific reactive site that is the prevalent antigen exposed when hexons are assembled in virions.

Question 6

Cellular transformation

Answer 6

All of the adenoviruses are transforming in tissue culture, but only viruses from groups A and B form tumors when injected in large amounts into newborn hamsters, rats, and mice.

No association with human tumors has been demonstrated!!!!!!!

Question 7

Epidemiology of adenovirus

Answer 7

Humans only

Infect all ages (particularly common in children).

Person-to-person spread in respiratory and ocular secretions is the most common mode of transmission.

Among children, fecal-oral transmission is important.

Dissemination in swimming pools has been implicated in epidemics of pharyngoconjunctival fever and conjunctivitis.

Adenoviruses continue to be excreted in feces (including from patients with respiratory and ocular infections) for days or, in rare cases, years after an initial infection, but serotypes 40 and 41, which cause diarrhea, are the only types clearly transmitted by the oral-fecal route.

Chronic viral shedding also occurs from the respiratory tract, but fecal shedding is more intense and prolonged.

Question 8

Serotype 4 of adenovirus

Answer 8

commonly causes acute respiratory disease (ARD) in military recruits but rarely produces clinical infections in civilians (this behavior is without parallel; its explanation is unknown). ARD in military recruits is largely confined to the cooler months. ARD can be transmitted experimentally by oral inoculation of infectious respiratory secretions.

Question 9

Are most adenovirus infections symptomatic?

Answer 9

NO!

Most adenovirus infections are asymptomatic.

Question 10

What percent of all respiratory infections in children under 5 years of age are due to adenoviruses?

Answer 10

5-10%

3rd most common after respiratory syncytial virus and parainfluenza virus

Question 11

What does isolating adenovirus mean for different body parts? (intenstine, throat, eye, etc)

Answer 11

Recovery from the eye, genital tract, lung, or brain is diagnostic;

From the throat of a patient with respiratory disease, suggestive

From feces, ambiguous, because adenoviruses are shed in large numbers and for long periods of time in the feces, particularly in children with persistent infections of tonsils and adenoids.

Question 12

Gene therapy with viral vectors

Answer 12

Offers great promise for the treatment of a variety of diseases. Virtually any virus that is or can be made defective has the potential to be useful for gene therapy.

Question 13

What are the three viruses currently most frequently used in gene therapy experiments? What should you consider when picking the vector?

Answer 13

retroviruses, adenoviruses, and adeno-associated virus (AAV)

In choosing a virus as an appropriate vector, questions that must be considered include the target cells and what viral receptors they express, the size of the DNA fragment to be introduced, the length of time that the transduced gene should be expressed, and the effects of the transducing virus on the host.

Question 14

What diseases are gene therapy useful for?

Answer 14

Inborn genetic and acquired diseases.

Acquired diseases can result from mutations (e.g., cancer) or infection (e.g., HIV).

In addition to gene-replacement thereapy, viral vectors offer promise for treatment of cancers and as vaccine agents.

Question 15

Retroviruses and how to use them for gene therapy

Answer 15

Enveloped and have an RNA genome that is converted to DNA by reverse transcription after infection.

The DNA integrates somewhat randomly, although predominantly into specific regions.

Packaging of viral vectors can be done using transient transfection of plasmids that encode the necessary functions, including capsid proteins and the protein of interest.

Only the long terminal repeats, the sequence that encodes the signal to package the genomic RNA within the virion, and sequences required for initiation of reverse transcription of the viral RNA need be retained in the virus vector.

Question 16

Advantages and disadvantages of using retroviruses for gene therapy

Answer 16

Advantages of retroviruses:

1. approximately 8 kb of foreign DNA can be used
2. stable expression of the transduced gene because of proviral integration in host DNA

Problems that arise from the use of retroviruses:

1. relatively small amounts of virus obtained during growth
2. virions are not very stable
3. non-mitotic cells are not transduced except by HIV vectors (which have a short triple-stranded region that is unique to lentiviruses and promotes viral DNA nuclear uptake)

Question 17

Adeno-associated virus (AAV)

Answer 17

Parvovirus with a naked capsid and a single-stranded DNA genome

Dependent for growth on viral gene products of adenovirus

In the presence of helper virus, wild-type AAV integrates efficiently into a chromosomal region devoid of gene sequences. In the absence of helper function, the wild-type virus may, when infecting at very high multiplicity, have its DNA converted to double-stranded by host proteins. The integrated virus can be rescued for growth upon superinfection with adenovirus.

Question 18

How to make vectors for AAV

Answer 18

AAV transducing vectors are constructed by replacing the viral coding sequence with foreign DNA while retaining the ends of the viral chromosome to provide origins of replication and the packaging sequence. The necessary viral functions for replication and capsid formation are provided in trans.

Question 19

Advantages and disadvantages of using AAV

Answer 19

Advantages in the use of AAV:

1. high viral titers can be obtained
2. the virions are very stable
3. approximately 5 kb of foreign DNA can be introduced
4. AAV has not been shown to cause clinical symptoms
5. coinfection with adenovirus and AAV leads to relatively site-specific integration of AAV DNA, so reinfection with a helper virus can lead to renewed AAV replication even after prolonged time periods

Disadvantages

1. the viral chromosome integrates, although inefficiently, into host DNA, a potentially mutagenic event
2. second strand DNA synthesis, gene expression, and integration are typically very slow in the absence of helper virus functions
3. the small size of the genome means that large genes and multiple genes cannot generally be introduced into the vector
4. in non-mitotic cells, transduction may be inefficient in the absence of adenovirus helper function

Question 20

Adenovirus vectors

Answer 20

Most adenovirus transducing vectors have the E1 region, which can be transforming, deleted and are complemented by growth in cells lines that provide E1 functions. With the deletion of the E1 region, the virus replicates very inefficiently in the absence of complementation.

Question 21

1st generation vs helper-dependent adenovirus vectors

Answer 21

First-generation adenovirus vectors have been intensively studied as gene therapy vectors and have substantial advantages. However, a strong inflammatory response occurs with the use of first-generation of vectors that leads to a strong adaptive immune response, precluding the effective reuse of the vector for gene replacement therapy, and shutoff of expression of genes contained within the viral vector.

Helper-dependent adenovirus vectors show considerable promise for long-term therapy, but are much more difficult to make and purify than are first-generation vectors.

Question 22

Advantages and disadvantages of adenovirus gene therapy

Answer 22

Advantages:

1. very high titers can be obtained
2. virions are fairly stable
3. the viral chromosome does not efficiently integrate
4. non-mitotic cells are efficiently transduced
5. less than 1 to greater than 30 kb of foreign DNA can be inserted, depending on the vector used
6. adenoviruses have the potential to be made to selectively replicate in, and kill, tumor cells

Disadvantages
1. adenovirus vectors elicit a strong inflammatory response (note that this is also a potential advantage: when targeting tumor cells or when used as a vaccine agent, the inflammatory response can be helpful); together with the fact that 60-90% of adult humans are seropositive to Ad5, the adenovirus most commonly used as a vector, this makes repeated use problematic

2. most of the adenovirus vectors currently in use require that, for virus growth, most of the adenovirus genes be retained in the vector; the presence of certain adenovirus genes leads to induction of an inflammatory response to the vector
3. expression of the transduced gene will be transient in mitotic cells
4. the death of a healthy patient, due to injection of an extremely high level of an adenoviral vector, in a gene therapy trial

Question 23

How is pH involved in the uptake of adenovirus (in a normal infection, not gene therapy)

Answer 23

Decreased pH allows for the virus to escape the endosome after receptor-mediated endocytosis

Question 24

Treatment of adenovirus

Answer 24

We don’t really

but maybe cydofovir and ribavirin