What Diseases Do Adeno-Associated Viruses Cause?

The party line about adeno-associated viruses is that they are "nonpathogenic." However, this claim is based on very limited investigation. Lack of acute symptoms of infection doesn't mean that they are harmless. It has been known since the early 1970s that AAVs cause latent infections by integrating into the cellular DNA, from which they can be reactivated by helper viruses including adenoviruses and herpesviruses, which are ubiquitous. Although up to 70% of adults are seropositive for AAV, there was virtually no interest in the effects of AAV integration and viral proteins until after AAVs began to be developed as potential gene therapy vectors.

EXAMPLE: "Despite its spectacular achievements, the first successful form of gene therapy will for now be used only as a last resort due to the risk of cancer. And that's not the only bad news. The latest studies suggest that some other gene therapies using different viruses might also trigger cancer. The Necker hospital in Paris pioneered the use of gene therapy to cure the severe inherited immune disease called X-SCID. But two of the 11 boys treated have developed leukemia. Both these cases appear to be due to the corrective gene being inserted near another gene called Lmo2, which helps control cell growth and can contribute to cancer if turned on at the wrong time (New Scientist, 25 January, p 12). Calculations by Christof von Kalle of the University of Cincinnati College of Medicine in Ohio suggest that rather than being an extraordinary coincidence the engineered mouse retrovirus used to deliver the gene in the French study may insert it near Lmo2 about once per 100,000 insertions. Since millions of bone marrow cells are modified and returned to the boys, such insertions may crop up in most if not all of the patients." (Cancer risk clouds gene cures. New Scientist March 15, 2003; Gene therapy 'caused T-cell leukemia." By Jo Lyford. The Scientist, Oct. 20, 2003; a third child later developed leukemia: January 2005, third adverse event in Paris! By Sandro Rusconi. Swiss Gene Therapy Web Page.) Although AAV was not used in this therapy, the principle is the same. And, what they do not mention in these articles is that the same thing may happen in natural infections - but, despite foreboding evidence, there has been little or no investigation before the establishment rushed into gene therapy.

LMO2-Associated Clonal T Cell Proliferation in Two Patients after Gene Therapy for SCID-X1. S Hacein-Bey-Abina, C Von Kalle, M. Schmidt, MP McCormack, N Wulffraat, P Leboulch, A Lim, C S Osborne, R Pawliuk, E Morillon, R Sorensen, A Forster, P Fraser, J I Cohen, G de Saint Basile, I Alexander, U Wintergerst, T Frebourg, A. Aurias, D Stoppa-Lyonnet, S Romana, I Radford-Weiss, F Gross, F Valensi, E Delabesse, E Macintyre, F. Sigaux, J Soulier, LE Leiva, M Wissler, C Prinz, TH Rabbitts, F Le Deist, A Fischer, M Cavazzana-Calvo. Science 2003 October 17;302(5644):415-419.

The Rep68 protein of adeno-associated virus type 2 stimulates expression of the platelet-derived growth factor B c-sis proto-oncogene. RS Wonderling, RA Owens. J Virol 1996 Jul;70(7):4783-4786. "The inappropriate expression of PDGF genes and their mitogenic products has been linked to several proliferative disorders such as fibrosis, atherosclerosis, and neoplasia. The ability of Rep68 to upregulate the c-sis proto-oncogene may imply a role for wild-type AAV in these diseases." Expression of several other genes to which Rep68 bound was not seen; however, some disorders are caused by reduced expression rather than increased expression of genes.

In a later study, Wonderling and Owens found 18 additional binding sites for AAV Rep68 protein (Binding sites for adeno-associated virus Rep proteins within the human genome. RS Wonderling, RA Owens. J Virol 1997 Mar;71(3):2528-2534).

Sites of recombinant adeno-associated virus integration. ED Rivadeneira, NC Popescu, DB Zimonjic, GS Cheng, PJ Nelson, MD Ross, JA DiPaolo, ME Klotman. Int J Oncol 1998 Apr;12(4):805-810. "Although recombinant AAVs, containing only the inverted terminal repeats of wild-type virus, can integrate efficiently into the host genome, specificity for the AAVS1 site appears to be lost.... Analysis of Southern blots indicated that none of twenty-six cell clones generated after infection with either one of the recombinant AAVs demonstrated integration within the AAVS1 locus on chromo-some 19. Analysis of five of the cell lines by fluorescent chromosome in situ hybridization confirmed the loss of chromosome 19 specificity. Each integration site mapped near a known fragile site and/or location of a proto-oncogene or growth regulatory gene. Retention of site-specific integration of wild-type AAV will require the inclusion of additional AAV-specific sequences within the recombinant vectors."

Adeno-associated virus site-specific integration and AAVS1 disruption. H Hamilton, J Gomos, KI Berns, E Falck-Pedersen. J Virol 2004 Aug;78(15):7874-7882. "Although maximal integration efficiency approached 40% of clonal cell lines (essentially 50% of infected cells), over 80% of cell lines contained a genomic disruption at the AAVS1 integration locus on chromosome 19 ( approximately 100% of infected cells)."

Large-scale analysis of adeno-associated virus vector integration sites in normal human cells. DG Miller, GD Trobridge, LM Petek, MA Jacobs, R Kaul, DW Russell. J Virology 2005 Sep;79(17):11434-11442. "[A]n extensive evaluation of adeno-associated virus (AAV) vector integration sites has not been completed, despite the ongoing use of AAV vectors in clinical trials." The authors have identified 977 integration junctions. "AAV vectors had a 4.8-fold preference for integration in CpG islands, which did not extend into surrounding regions... There was also a significant preference for integration in CpG islands (4.03% versus 0.84% for calculated random integrants), which typically act as transcriptional control elements for nearby genes. Surprisingly, some of our results were similar to those of murine leukemia virus vectors, where integrations also occurred preferentially near transcription start sites and CpG islands, with only a slight overall preference for genes. Thus, despite the distinct life cycles of these viruses, they may share aspects of integration site selection."

Characterization of adeno-associated virus genomes isolated from human tissues. BC Schnepp, RL Jensen, CL Chen, PR Johnson, KR Clark. J Virol 2005 Dec;79(23):14793-14803. Infection with wild-type adeno-associated virus (AAV) is common in humans, but very little is known about the in vivo biology of AAV. On a molecular level, it has been shown in cultured cells that AAV integrates in a site-specific manner on human chromosome 19, but this has never been demonstrated directly in infected human tissues. To that end, we tested 175 tissue samples for the presence of AAV DNA, and when present, examined the specific form of the viral DNA. AAV was detected in 7 of 101 tonsil-adenoid samples and in 2 of 74 other tissue samples (spleen and lung). In these nine samples, we were unable to detect AAV integration in the AAVS1 locus using a sensitive PCR assay designed to amplify specific viral-cellular DNA junctions. Additionally, we used a second complementary assay, linear amplification-mediated-PCR (LAM-PCR) to widen our search for integration events.... Given these data, we entertained the possibility that instead of integrated forms, AAV genomes were present as extrachromosomal forms. We used a novel amplification assay (linear rolling-circle amplification) to show that the majority of wild-type AAV DNA existed as circular double-stranded episomes in our tissues. Thus, following naturally acquired infection, AAV DNA can persist mainly as circular episomes in human tissues. These findings are consistent with the circular episomal forms of recombinant AAV vectors that have been isolated and characterized from in vivo transduced tissues."

AAV and skeletal muscle gene TNNT1

Adeno-associated virus site-specifically integrates into a muscle-specific DNA region. N Dutheil, F Shi, T Dupressoir, RM Linden. Proc Natl Acad Sci USA 2000 Apr;97(9):4862-4866. "These data indicate that integration of AAV into the AAVS1 locus can lead to rearrangements of the TNNT1 gene."

AAV and skin

Ubiquitous human adeno-associated virus type 2 autonomously replicates in differentiating keratinocytes of a normal skin model. C Meyers, M Mane, N Kokorina, S Alam, PL Hermonat. Virology 2000 Jul 5;272(2):338-346. "Since its discovery in 1966, adeno-associated virus type 2 (AAV) has been described as a helper-dependent parvovirus. However, in this study we demonstrate that AAV undergoes its complete life cycle, devoid of helper viruses or genotoxic agents, in the organotypic epithelial raft culture system, a model of normal skin.... Additionally, dosage dependent histologic changes, some of which might be interpreted as cytopathology, were induced in the AAV-infected epithelial tissues."

Adeno-associated virus expresses transgenes in hair follicles and epidermis. UR Hengge, A Mirmohammadsadegh. Mol Ther 2000 Sep;2(3):188-194. AAV "efficiently targets hair follicle epithelial cells."

AAV in bone marrow progenitor cells

Detection of adeno-associated virus type 2 in sorted human bone marrow progenitor cells. RJ Anderson, G Galatowicz, ID Macdonald, MW Lowdell, TJ Corbett, HG Prentice. Exp Hematol 1997 Mar;25(3):256-262. Integrated virus was found in 18/106 bone marrow samples; in 0/3 CD34+ cells, but 3/3 CD3+ cells from PCR-positive individuals.

DNA of adeno-associated virus (AAV) in testicular tissue and in abnormal semen samples. K Erles, V Rohde, M Thaele, S Roth, L Edler, JR Schlehofer. Hum Reprod 2001 Nov;16(11):2333-2337. And: Adeno-associated virus infection linked to male infertility. (News) Medscape - Reuters Health 2001 Oct 30.

Evidence of chromosomal integration of AAV DNA in human testis tissue. S Mehrle, V Rohde, JR Schlehofer. Virus Genes 2004 Jan;28(1):61-69. "These data demonstrate that AAV DNA can integrate also after natural infection, and that integration occurs within the AAVS1 region, at least in some cases."

AAV Infection During Pregnancy Causes Spontaneous Abortions and Trophoblastic Disease

Detection of adeno-associated virus DNA in human genital tissue and in material from spontaneous abortion. E Tobiasch, M Rabreau, K Geletneky, S Laruë-Charlus, F Severin, N Becker, JR Schlehofer. J Med Virol 1994 Oct;44(2):215-222. "Using the polymerase chain reaction (PCR) AAV type 2 DNA was amplified in histological sections of 19 of 30 biopsies of the uterine mucosa. In addition, AAV-2 DNA was detected in abortion material during the first trimester of pregnancy (12/30 cases were positive) but not in material of abortion from the second or third trimester (9 cases). Whereas in tissues from the uterus AAV DNA was found only by PCR, large amounts of viral DNA were detectable by Southern blot analysis in abortion material. In situ hybridization revealed DNA of AAV to be present in the villous moiety (trophoblast) of the placenta but not in the embryo or decidua. in the same cells, AAV proteins (including the replication-associated rep proteins) were detected by immunofluorescence analysis. These results suggest (1) that AAV infects the uterine mucosa (possibly persistently) and (2) that it can replicate in trophoblast cells. This might disturb placenta development and may play a role in early miscarriage."

Evidence for infection of the human embryo with adeno-associated virus in pregnancy. T Burguete, M Rabreau, M Fontanges-Darriet, E Roset, HD Hager, A Koppel, P Bischof, JR Schlehofer. Hum Reprod 1999 Sep;14(9):2396-2401. "In mothers with AAV DNA-positive amnion fluids, premature amniorrhexis and premature labour occurred significantly more frequently (P<0.001)."

Adeno-associated virus DNA in human gestational trophoblastic disease. K Kiehl, JR Schlehofer, R Schultz, M Zugaib, E Armbruster-Moraes. Placenta 2002 May;23(5):410-415. "AAV DNA was found in 43 samples (28/49 hydatiform moles, 4/14 choriocarcinomas, 11/15 miscarriage material). These findings confirm AAV infection of embryo-derived tissue in humans and further suggest a role of AAV in miscarriage and trophoblastic disease."

Adeno-associated virus-2 (AAV-2) causes trophoblast dysfunction, and placental AAV-2 infection is associated with preeclampsia. F Arechavaleta-Velasco, Y Ma, J Zhang, CM McGrath, S Parry. Am J Pathol 2006 Jun;168(6):1951-1959. "We determined that transformed extravillous trophoblast (HTR-8/SVneo) cells were susceptible to AAV-2 infection in the presence or absence of adenovirus, which provides helper function for AAV-2 replication, and that AAV-2 infection reduced invasion of HTR-8/SVneo cells through an extracellular matrix before cytopathic effects were detected. In a case-control study, AAV-2 DNA was found more frequently in trophoblast cells from cases of severe preeclampsia (22/40) than from normal term deliveries (5/27, P = 0.002)."

Adverse reproductive outcomes in urban women with adeno-associated virus-2 infections in early pregnancy. F Arechavaleta-Velasco, L Gomez, Y Ma, J Zhao, CM McGrath, MD Sammel, DB Nelson, S Parry. Hum Reprod 2008 Jan;23(1):29-36. "First trimester maternal IgM seropositivity was 5.6 times more prevalent among pre-eclampsia/IUGR/stillbirth cases (P = 0.0004) and 7.6 times more prevalent among preterm deliveries (P < 0.0001) than among controls. CMV and adenovirus IgM antibodies and chronic AAV-2 infections (IgG seropositivity) were not associated with adverse pregnancy outcomes. CONCLUSIONS Primary or reactivated AAV-2 infection (maternal IgM seropositivity) early in pregnancy was associated with adverse reproductive outcomes associated with placental dysfunction, including pre-eclampsia, stillbirth and spontaneous preterm delivery."

AAV and HPV

There were earlier reports that AAV infection might protect against cervical cancer by interfering with human papillomavirus infection. However, epidemiological studies have not confirmed this. (Adeno-associated virus and development of cervical neoplasia. HD Strickler, R Viscidi, C Escoffery, C Rattray, KL Kotloff, J Goldberg, A Manns, C Rabkin, R Daniel, B Hanchard, C Brown, M Hutchinson, D Zanizer, J Palefsky, RD Burk, B Cranston, B Clayman, KV Shah. J Med Virol 1999 Sep;59(1):60-65; and: Evaluation of the possible protective role of adeno-associated virus type 2 infection in HPV-associated premalignant disease of the cervix. KO Odunsi, CC van Ee, TS Ganesan, AN Shelling. Gynecol Oncol 2000 Sep;78(3 Pt 1):342-345.)

Correlation of human papillomavirus and adeno-associated virus 2 infection in cytology with Korean women. J Hyun-Sun, C Heung-Jae, K Byung-Hun, C Young-Lae, N Gye-Hyun, C Ho-Sun, K Chong-Kook, H Byoung-Don, B Su-Mi, A Woong-Shick. Int J Gynecol Cancer 2006 Mar-Apr;16(2):604-609. "The correlation between AAV 2 and HPV 16 was statistically significant in normal and CIN I/II group only, and AAV 2 and HPV 16 and/or HPV 18 showed no correlation. Therefore, the correlation between AAV and HPV were not statistically significant. These data support the previous reports that AAV might not be associated with cervical tumorigenesis."

Doubletalk about AAV

Virus-mediated killing of cells that lack p53 activity. K Raj, P Ogston, P Beard. Nature 2001 Aug 30;412(6850):914-917; and: Virus selectively kills p53-deficient cancer cells. Medscape - Reuters Health 2001 Aug 29. The study indicates that AAV DNA "elicits in cells a DNA damage response," which should not be surprising considering that AAV integrates into cellular DNA. However, in the news report the authors state: "Whereas further work will elucidate what kind of DNA damage AAV DNA is mimicking [sic!] and details of which pathway is being triggered, the immediate importance of these findings is the introduction of the principle of using viruses to efficiently deliver DNA with unusual or modified structures into cells to eliminate those cells that lack p53 activity." "Mimicking" DNA damage?? That is a peculiar choice of words. It suggests not only a preference for discovering a new use for AAV in cancer therapy over their role in the causation of disease, but also a desire to overlook those kind of implications. And no doubt it is because a new cancer therapy is more likely to win its discoverer(s) one of those much-sought trips to Stockholm than is a contribution to the prevention of cancer.

It is possible that this simply won't work. Offner et al. found that "malignant cells may leave the primary carcinoma at an early stage of its development when p53 is not mutated yet, and targeting mutated p53 is unlikely to be a successful strategy to detect and to eliminate these potential precursors of subsequent overt metastasis." (P53 gene mutations are not required for early dissemination of cancer cells. S Offner, W Schnaus, K Witter, GB Baretton, G Schlimok, B Passlick, G Riethmuller, K Pantel. PNAS 1999 Jun;96:6942-6946.) It has also been found that "Isolated tumor cells in bone marrow predict reduced survival in node-negative non-small cell lung cancer" (B Passlick, B Kubuschok, JR Izbicki, O Thetter, K Pantel. Ann Thoracic Surg 1999 Dec;68(6):2053-2058). So once again, energies are being misdirected on a wild goose chase, while the implications for etiology and prevention are ignored.

Inhibition of S-phase progession by adeno-associated virus Rep78 protein is mediated by hypophosphorylated pRb. P Saudan, J Vlach, P Beard. EMBO J 2000;19(16):4351-4361. In cells infected with an AAV vector which lacks the Rep gene, the adenovirus E4orf6 protein also arrests cells in S-phase, with reversibility by cyclin A. "Hence, in a wild-type AAV infection, Rep78, which we have shown to affect the cells similarly, could promote the establishment of a latent infection."

[Note that the adenovirus E4orf6 protein has since been implicated in "hit-and-run" transformation!] "Hit-and-run" transformation by adenovirus oncogenes. M Nevels, B Tauber, T Spruss, H Wolf, T Dobner. J Virol 2001 Apr;75(7):3089-3094.) Is AAV rep78 protein similar to adenovirus E4orf6 protein in this respect as well?

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cast 01-27-08