Construction of adeno-associated virus packaging plasmids and cells that directly select for AAV helper functions

Recombinant adeno-associated virus type 2 (rAAV) has promise for use as a gene therapy vector. Potential problems in the production of rAAV stocks are both the limited amount of recombinant virus that is produced by traditional methods and the possibility of wild-type replication competent adeno-associated virus (wtAAV) contamination. The presence of these contaminants is largely dependent upon the helper plasmid used. Whilst wtAAV is not a pathogen, the presence of these contaminants is undesirable as they may affect experiments concerning the biology of rAAV. Additionally as protocols using rAAV with altered tropism are becoming more prevalent, it is important that no recombination be permitted that may cause the creation of a replication competent AAV with modified (targeting) capsids. Many experimental protocols require the generation of large amounts of high titre rAAV stocks. We describe the production of several AAV helper plasmids and cell lines designed to achieve this goal. These plasmids possess split AAV rep and cap genes to eliminate the production of wtAAV and they possess a selection mechanism which is operatively linked to expression from the AAV cap gene. This allows positive selection of those cells expressing the highest level of the structural capsid proteins and therefore those cells which yield the highest amount of rAAV.     

Update on the prevalence of serum antibodies (IgG and IgM) to adeno-associated virus (AAV).

In view of presumed non-pathogenicity, tumor suppressive properties, and site-specific integration of the viral genome the human parvovirus, adeno-associated virus (AAV) has gained great interest as a gene transduction vector. Data on the seroprevalence of antibodies to AAV vary between reports, probably due to the different serological methods used. In order to understand better the immune response to AAV during natural infection, sera from different age groups and various geographical regions were compared for AAV antibodies using an ELISA. The data show that the prevalence of antibodies to AAV is similar in Europe (Germany, France, and Switzerland), Brazil, and Japan, indicating worldwide infection. It was confirmed that infection takes place during childhood. However, declining seropositivity thereafter and a second increase of seropositivity after 30 years of age suggests reinfection or reactivation of latent virus in particular as the prevalence of IgM antibodies in adults is relatively high. Furthermore, pregnant women were found to be significantly more frequently seropositive than non-pregnant controls, hinting at a reactivation of persistent AAV (up to 80% of women carry AAV in genital tissue) in specific hormonal conditions, e.g., pregnancy. Cross-reaction of serum antibodies with the different AAV types (defined by complement fixation) was observed by ELISA and neutralization tests confirming earlier results. The results suggest an unstable AAV antibody response allowing lifelong reinfection or reactivation of persisting virus possibly due to partial immunotolerance after an infection in utero, at delivery or during early infancy.     

From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy

Gene transfer vectors based on the human adeno-associated virus serotype 2 (AAV-2) have been developed and tested in pre-clinical studies for almost 20 years, and are currently being evaluated in clinical trials. So far, all these studies have provided evidence that AAV-2 vectors possess many properties making them very attractive for therapeutic gene delivery to humans, such as a lack of pathogenicity or toxicity, and the ability to confer long-term gene expression. However, there is concern that two restrictions of AAV-2 vectors might limit their clinical use in humans. First, these vectors are rather inefficient at transducing some cells of therapeutic interest, such as liver and muscle cells. Second, gene transfer might be hampered by neutralizing anti-AAV-2 antibodies, which are highly prevalent in the human population. In efforts to overcome both limitations, an increasing number of researchers are now focusing on the seven other naturally occurring serotypes of AAV (AAV-1 and AAV-3 to -8), which are structurally and functionally different from AAV-2. To this end, several strategies have been devised to cross-package an AAV-2 vector genome into the capsids of the other AAV serotypes, resulting in a new generation of "pseudotyped" AAV vectors. In vitro and in vivo, these novel vectors were shown to have a host range different from AAV-2, and to escape the anti-AAV-2 immune response, thus underscoring the great potential of this approach. Here the biology of the eight AAV serotypes is summarized, existing technology for pseudotyped AAV vector production is described, initial results from pre-clinical evaluation of the vectors are reviewed, and finally, the prospects of these promising novel tools for human gene therapy are discussed.     

High prevalence of adeno-associated virus (AAV) type 2 rep DNA in cervical materials: AAV may be sexually transmitted

Adeno-associated virus (AAV) is a human parvovirus that in laboratory and animal models has the ability to supress the oncogenic phenotype of a variety of viruses and cellular derived oncogenes. The inhibitory effects of AAV have been mapped to its rap gene (Rep 78 protein). Furthermore, seroepidemiologic data indicate that AAV infection is linked to reduced cervical cancer rates in humans. Because of AAV's inverse relationship with cervical cancer, we attempted to identify AAV rep sequences within DNA derived from cervical brushings taken from nondiseased middle class patients at a Little Rock clinic. Polymerase chain reaction (PCR) amplification was carried out with primers designed to amplify a specific segment of the endogenous human -globin gene or the AAV rep gene. Of those cervical samples that were positive for -globin DNA, 50% were also found to be positive for AAV rep DNA when analyzed by either ethidium bromide staining or dot-blot hybridization with an internal probe. These data strongly suggest that AAV is commonly carried in the genital region and further raise the possibility that AAV can be sexually transmitted.     

DNA of adeno-associated virus (AAV) in testicular tissue and in abnormal semen samples.

BACKGROUND: Human genital tissues, including spermatozoa, have been found to be frequently infected with the helper-virus dependent parvovirus, adeno-associated virus (AAV). METHODS: To assess the role of AAV infection in disorders of the male reproductive system, semen samples from 95 men (including 73 men attending a fertility programme) and testicular samples from patients with azoospermia (n = 38) or prostate cancer (n = 8) were analysed using polymerase chain reaction for the presence of AAV DNA. Semen quality was assessed according to World Health Organization guidelines and the grade of atrophy of testicular biopsies was determined histomorphologically. RESULTS: AAV DNA was detected in 38% (28/73) of ejaculates from men with abnormal semen analyses (oligoasthenozoospermia or asthenozoospermia) and in 4.6% of normal semen samples (1/22, P = 0.003). DNA from AAV helper-viruses (human papillomaviruses, cytomegalovirus) was detected at similar frequencies in normal and abnormal semen samples. In testes, AAV DNA was detected in 10 out of 38 biopsies from infertile men (26%), and in 2 out of 8 orchidectomy samples. CONCLUSION: The data show an increased incidence of AAV infection with abnormal semen analysis. Detection of AAV DNA in the testes might point to a role for AAV infection in male infertility, possibly by interfering with spermatozoa development.     

Detection of adeno-associated virus (AAV)-specific nucleotide sequences in DNA isolated from latently infected Detroit 6 cells.

Detroit 6 cells exposed to 250 TCID₅₀/cell of adeno-associated virus (AAV)-2 retained the ability to produce AAV antigens and infectious virus upon subsequent exposure to helper adenovirus type 2 for many passages. The increased rate of annealing of ³²P-labeled AAV-DNA in the presence of cellular DNA from two such latently infected clones indicated the presence of three to five AAV genome equivalents per diploid amount of cell DNA.     

Avian adeno-associated virus (AAAV) and fowl adenoviruses (FAV): Studies of viral interactions in chicken cell cultures

As the growth kinetics of avian adeno-associated virus (AAAV) in chicken cells demonstrate, the three serotypes of fowl adenovirus (FAV), FAV -1, -5 and -8, provide complete helper activity for the production of infectious AAAV. Under one step conditions, the growth cycle of AAAV in primary chicken kidney cell (CKC) cultures is characterised by an eclipse phase of 8 hours and an exponential increase of the virus infectivity by 4 to 5 logs until 24 hours post-adsorption (p.a.). These growth characteristics do not depend on the serotype of FAV used as helper. In chicken embryo fibroblast (CEF) cultures the eclipse phase is prolonged to 12 hours p.a. and the virus infectivity increases only by 2 logs. In addition, the low efficiency of plating of FAV -1 in this cell system does not allow one step growth curves for AAAV. In CKC and CEF cultures coinfected with FAV and AAAV the multiplication of helper FAV is reduced. The degree of growth inhibition depends on the AAV multiplicity used. Sequential infection of CKC cultures with FAV -1 and AAAV modifies the AAAV growth cycle, i.e. there is a time reduction of the eclipse phase and a decrease of the virus yield. Infectious AAAV was determined by an indirect immunofluorescence assay and infectious FAV by a plaque assay.     

Studies on the defectiveness of adeno-associated virus (AAV). II. Effect of growth in CV-1 cells on the replication of adeno-associated virus.

The mechanisms by which helper viruses can promote AAV macromolecular synthesis have been investigated. The third system used was a continuous line of African green monkey kidney cells (CV-1) which is permissive for AAV when a simian adenovirus helper is used. When HSV was used as a helper, no AAV DNA, structural proteins, or IF antigens were detected, indicating the absence of HSV helper activity. HSV production and plaqueing in CV-1 cells were found to be comparable to those seen in Hep-2 cells (an epidermoid cancer line) and Vero cells (another line of African green monkey kidney cells). In both of these cell lines, HSV expresses its AAV helper functions. It was also observed that AAV had no effect on HSV replication in either Vero or CV-1 cells. Comparison of HSV protein production in CV-1 and Hep-2 cells demonstrated the absence of HSV proteins ICPO and ICP46 in the CV-1 line following the release of a cycloheximide block.     

Temporal acceleration of the human papillomavirus life cycle by adeno-associated virus (AAV) type 2 superinfection in natural host tissue

Epidemiologically, certain human papillomaviruses are positively associated with cervical cancer, while adeno-associated viruses (AAV-2) are negatively associated with this same cancer. Both HPV and AAV productively replicate in differentiating keratinocytes of the skin and interact with each other. However, AAV has a relatively fast life cycle, generating infectious progeny by the third to fourth day of an organotypic epithelial raft culture. In contrast, HPV is slow, generating infectious progeny only after 10-12 days. As earlier studies indicated that these two skin-tropic virus types significantly affect each other's life cycle, we investigated if the temporal kinetics of the slow HPV life cycle was affected by the fast AAV in raft cultures. Here it is shown that the presence of AAV-2 at a variety of multiplicities of infection (m.o.i.) resulted in early onset HPV-31b DNA replication. Using plasmids which each expressed only one of the four rep proteins, an enhancement affect was seen for all four rep proteins of AAV, with Rep40 having the highest activity. Furthermore, AAV (m.o.i. of 5) also resulted in a temporally accelerated production of HPV infectious units, seen as early as Day 4, with high levels of viral progeny being produced by Day 6.5. Like earlier studies at Day 12, histological differences were seen at Day 6.5 between AAV-infected and mock-infected HPV/rafts. These data suggest that under specific conditions the AAV rep trans-factors can positively regulate HPV gene expression in addition to the usual negative regulation that has been consistently observed by the rep proteins. These data also suggest that AAV has a significant effect upon the temporal kinetics of the HPV life cycle in natural host tissue. However, it is unclear if or how this AAV-induced fast HPV life cycle mechanistically correlates with lower rates of HPV-associated cervical disease.     

Studies on the defectiveness of adeno-associated virus (AAV). I. Effects of phosphonoacetic acid and 2-deoxy-D-glucose on the replication of AAV.

Adeno-associated viruses (AAV) are defective parvoviruses which require the presence of a helper virus, either an adenovirus or a herpesvirus, to initiate their replication cycle. The mechanisms by which these helper viruses can promote AAV macromolecular synthesis have been investigated in systems where the replication of the helper virus was altered. In the first system, phosphonoacetic acid (PAA), a specific inhibitor of herpesvirus-coded DNA polymerase, was used in AAV-herpes simplex virus (HSV) coinfections to determine what effect this drug would have on the replication of AAV. It was found that in the presence of increasing concentrations of PAA, the synthesis of AAV DNA, structural proteins, and immunofluorescent (IF) capsid antigens was inhibited. However, when an adenovirus helper was used in place of HSV, this inhibition was not seen. It was also found that the addition of the drug 5 hr after infection was still effective in inhibiting AAV capsid antigen synthesis completely. This finding indicates that the PAA-sensitive event required by AAV occurs relatively late in the HSV cycle. Restoration of HSV DNA polymerase activity by reversal of a PAA block was not sufficient for initiating AAV replication. De novo protein synthesis was required also. In the second system, the effects of 2-deoxy-d-glucose (2DG), an inhibitor of protein glycosylation, on AAV replication were also examined. In AAV coinfections with HSV, increasing concentrations of 2DG inhibited the production of AAV IF capsid antigens. Conversely, production of AAV intracellular proteins was enhanced with increasing 2DG concentrations. Under these conditions the pattern of IF staining for the major AAV polypeptide was normal. Thus 2DG appears to interfere with the assembly of AAV proteins into a capsid configuration. In experiments with an adenovirus helper, 2DG was found to inhibit initiation of AAV replication through early adenovirus functions.     

Use of a BCG vaccine from a Soviet substrain for the immunotherapy of tumors caused by SA7 (C8) virus

The oncolytic and immunotherapeutic effect of the national BCG vaccine with respect to undifferentiated sarcoma of hamster induced by the highly oncogenic adenovirus of green monkeys SA7(C8) was studied. Preliminary data on significant positive effect of the vaccine inoculated before tumor formation were obtained. A single inoculation of the vaccine into tumors did not result in tumor regression in any of the cases.     

Use of a simple separation column in detection of immunoglobulin M antibody to Epstein-Barr virus.

The use of a new commercial separation column on whole test serum improved the method for detecting immunoglobulin M antibody to Epstein-Barr virus. The efficiency of this product in absorbing interfering immunoglobulin G was similar to that of another absorption product, staphylococcal protein A, but it has the advantage of being stable without refrigeration.     

Identification of a region of the HSV-1 genome with helper activity for AAV

A specific fragment of the herpes simplex virus type I genome has been shown to have the ability to provide helper activity for adeno-associated virus replication. Restriction endonuclease generated DNA fragments from either herpes viral DNA or recombinant plasmids were transfected into cultured cells. The cells were then infected with adeno-associated virus, and helper activity was detected by fluorescent antibody staining of the cells for adeno-associated virus infected antigens. The region with AAV helper activity is 1.75 kb in size and makes up only about 1% of the HSV-1 genome. It is located between map coordinates 0.316 and 0.327 on the prototype arrangement of the HSV-1 genome and is the first function to be mapped in this region.