Adeno-Associated Virus Vectors and Hematology

Abstract

GENE THERAPISTS have a special fondness for hematology. Initially, this was due to the many inherited diseases that could potentially be cured by ex vivo genetic modification of hematopoietic stem cells. Once the early murine leukemia virus (MLV) vectors were found to transduce only a small percentage of primate stem cells, the search began for other types of vectors that might work more efficiently, and adeno-associated virus (AAV) vectors were among the first alternatives to be investigated. Although a substantial amount of work with AAV vectors has occurred over the last several years, the bulk of evidence suggests that they transduce hematopoietic stem cells poorly, and that they will not replace retroviral vectors in stem cell gene transfer applications. Instead, it is applications involving the production of secreted proteins that now appear especially promising for AAV vectors, where they are delivered by direct injection rather than ex vivo cell manipulation. Recent animal studies have shown that AAV vectors encoding factor IX can lead to long-term, therapeutic clotting factor levels after in vivo administration, setting the stage for what may well be a cure for hemophilia B in the near future. Animal experiments have also shown that AAV vectors expressing erythropoietin can produce dramatic increases in hematocrit levels. Here we will review the role of AAV vectors in the field of hematology, including the basic biology of AAV, vector transduction properties, their use in hematopoietic cells, and their potential for delivering secreted proteins such as clotting factors and cytokines. BIOLOGY OF AAV AAV is a dependent parvovirus with an ≈4.7-kb single-stranded linear genome that contains two open reading frames (rep and cap). 1 The genome encodes 4 replication (Rep) proteins and 3 capsid proteins (VP1-3) (Fig 1). The inverted terminal repeats (ITRs) can pair to form characteristic T-shaped hairpins, which are the only sequences required in cis for replication and packaging. For a productive infection to occur, AAV requires coinfection with a helper virus such as adenovirus, which allows the viral genome to replicate episomally, and leads to synthesis of the AAV proteins. In the absence of helper virus, the AAV genome can integrate into the host cell chromosome, where it remains in a latent proviral state until infection with helper virus occurs, at which point the proviral genome excises and replicates, and a productive infection resumes. Several different serotypes of AAV have been identified by serological analysis, 2-5 and DNA sequencing has shown significant differences in their capsid genes that presumably account for the distinct serological profiles. 6-11 Antibodies against AAV types 1-3 and 5 are frequently found in human serum samples. 12-14 Virtually all the AAV vectors developed to date were based on AAV type 2, which has implications for gene therapy, because there is a high prevalence of neutralizing antibodies against this serotype in human populations. 14,15

CRISPR/Cas Applications in Myotonic Dystrophy: Expanding Opportunities

“…Consequentially, AAV-CRISPR/Cas9 can be introduced into patients only once in order to avoid the amplification of the adaptive immune response, thus limiting efficiency to a single dose of the treatment, which might be insufficient [80]. Whilst AAV2 is the best characterized and mostly used vector in clinical studies [76,81], antibodies against the AAV2 serotype are also the most common [82]. To overcome the presence of preexisting immunity against AAV and enable re-administration, researchers are currently using capsid gene shuffling to produce viral variants resistant to neutralizing antibodies [76,81].…”

Adeno‐associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo†‡

“…Even now, gene therapy efforts remain focused on gene addition strategies using full‐length complementary DNA (cDNA) cassettes for the mutated gene of interest, driven by promoter and enhancer sequences 1. Despite many advances, gene addition approaches with adeno‐associated virus (AAV) are limited by transient and unregulated expression,2 highly random integrations,3 transgene silencing,4 and increased mutagenic and oncogenic risks 5. Not all protein‐coding genes have open reading frames small enough to fit within the low coding capacity of AAV (4.7 kb), thus, this type of gene therapy is not applicable for all disorders 6…”

Adeno‐associated virus (AAV)‐mediated transduction of male germ line stem cells results in transgene transmission after germ cell transplantation

“…linear genome that can integrate in a site-specific manner into human chromosome 19 in replicating and nonreplicating cells (1,2). However, in the absence of the viral rep protein, integration occurs by nonhomologous recombination at random locations.…”