Thorough molecular configuration analysis of noncanonical AAV genomes in AAV vector preparations.

The unique palindromic inverted terminal repeats (ITRs) and single-stranded nature of adeno-associated virus (AAV) DNA are major hurdles to current sequencing technologies. Due to these characteristics, sequencing noncanonical AAV genomes present in AAV vector preparations remains challenging. To address this limitation, we developed thorough molecule configuration analysis of noncanonical AAV genomes (TMCA-AAV-seq). TMCA-AAV-seq takes advantage of the documented AAV packaging mechanism in which encapsidation initiates from its 3' ITR, for AAV-seq library construction. Any AAV genome with a 3' ITR is converted to a template suitable to adapter addition by a Bst DNA polymerase-mediated extension reaction. This extension reaction helps fix ITR heterogeneity in the AAV population and allows efficient adapter addition to even noncanonical AAV genomes. The resulting library maintains the original AAV genome configurations without introducing undesired changes. Subsequently, long-read sequencing can be performed by the Pacific Biosciences (PacBio) single-molecule, real-time (SMRT) sequencing technology platform. Finally, through comprehensive data analysis, we can recover canonical, noncanonical AAV DNA, and non-AAV vector DNA sequences, along with their molecular configurations. Our method is a robust tool for profiling thorough AAV-population genomes. TMCA-AAVseq can be further extended to all parvoviruses and their derivative vectors.

Subgenomic particles in rAAV vectors result from DNA lesion/break and non-homologous end joining of vector genomes.

Recombinant adeno-associated virus (rAAV) vectors have been developed for therapeutic treatment of genetic diseases. Current rAAV vectors administered to affected individuals often contain vector DNA-related contaminants. Here we present a thorough molecular analysis of the configuration of non-standard AAV genomes generated during rAAV production using single-molecule sequencing. In addition to the sub-vector genomic-size particles containing incomplete AAV genomes, our results showed that rAAV preparations were contaminated with multiple categories of subgenomic particles with a snapback genome (SBG) configuration or a vector genome with deletions. Through CRISPR and nuclease-based modeling in tissue culture cells, we identified that a potential mechanism leading to formation of non-canonical genome particles occurred through non-homologous end joining of fragmented vector genomes caused by genome lesions or DNA breaks present in the host cells. The results of this study advance our understanding of AAV vectors and provide new clues for improving vector efficiency and safety profiles for use in human gene therapy.

Satellite Subgenomic Particles Are Key Regulators of Adeno-Associated Virus Life Cycle.

Historically, adeno-associated virus (AAV)-defective interfering particles (DI) were known as abnormal virions arising from natural replication and encapsidation errors. Through single virion genome analysis, we revealed that a major category of DI particles contains a double-stranded DNA genome in a "snapback" configuration. The 5'- snapback genomes (SBGs) include the P5 promoters and partial rep gene sequences. The 3'-SBGs contains the capsid region. The molecular configuration of 5'-SBGs theoretically may allow double-stranded RNA transcription in their dimer configuration. Our studies demonstrated that 5-SBG regulated AAV rep expression and improved AAV packaging. In contrast, 3'-SBGs at its dimer configuration increased levels of cap protein. The generation and accumulation of 5'-SBGs and 3'-SBGs appears to be coordinated to balance the viral gene expression level. Therefore, the functions of 5'-SBGs and 3'-SBGs may help maximize the yield of AAV progenies. We postulate that AAV virus population behaved as a colony and utilizes its subgenomic particles to overcome the size limit of a viral genome and encodes additional essential functions.

Expression of stimulated by retinoic acid gene 8 (Stra8) and maturation of murine gonocytes and spermatogonia induced by retinoic acid in vitro.

Vitamin A deficiency in the mouse results in an arrest in the progression of undifferentiated spermatogonia to differentiating spermatogonia. The supplement of retinol to vitamin-A-deficient mice reinitiates spermatogenesis in a synchronous manner throughout the testes. It is unclear whether the effects of retinoids are the result of a direct action on germ cells or are indirectly mediated through Sertoli cells. The expression of Stimulated by retinoic acid gene 8 (Stra8), which is required for spermatogenesis, is directly related to the availability of retinoic acid (RA). Analysis of gene expression by microarrays revealed moderate levels of Stra8 transcript in gonocytes and high levels in A and B spermatogonia. Stra8 mRNA levels were greatly reduced or absent in germ cells once they entered meiosis. This study examined the effect of retinoic acid on cultured neonatal testes and isolated gonocytes/spermatogonia in vitro. THY1(+) and KIT(+) germ cells were isolated by magnetic-activated cell sorting from the testes of mice of different ages. Isolated germ cells were cultured and treated with either vehicle (ethanol) or RA without feeder cells. We found that 1) Stra8 is predominantly expressed in premeiotic germ cells, 2) RA stimulates gonocyte DNA replication and differentiation in cultured neonatal testes, 3) in the absence of feeder cells, RA directly induces the transition of undifferentiated spermatogonia to differentiating spermatogonia by stimulating Stra8 and Kit gene expression, 4) RA dramatically stimulates Stra8 expression in undifferentiated spermatogonia but has a lesser impact in differentiating spermatogonia, 5) endogenous Stra8 gene expression is higher in differentiating spermatogonia than in undifferentiated spermatogonia and could mediate the RA effects on spermatogonial maturation, and 6) RA stimulates a group of genes involved in the metabolism, storage, transport, and signaling of retinoids.

High-mobility group A1a protein regulates Ras/ERK signaling in MCF-7 human breast cancer cells.

High-mobility group (HMG) A1 proteins are gene regulatory factors whose overexpression is frequently observed in naturally occurring human cancers. The overexpression of transgenic HMGA1 proteins in cells results in neoplastic transformation and promotes progression to malignant cellular phenotypes. To understand the underlying molecular and biological events involved in these phenomena, we used oligonucleotide microarray analyses to generate an HMGA1a-induced expression profile for approximately 22,000 genes. This gene expression profile was generated using a well-characterized transgenic human MCF-7 mammary adenocarcinoma cell line in which overexpression of transgenic HMGA1 promotes a transition to a more malignant and metastatic phenotype. Microarray expression analyses, together with independent quantitative real-time reverse transcriptase polymerase chain reaction results, indicate that HMGA1a regulates genes involved in the Ras-extracellular signal-related kinase (Ras/ERK) mitogenic signaling pathway, including KIT ligand and caveolins 1 and 2. We also found that many cholesterol biosynthesis genes were decreased in cells overexpressing HMGA1a. Cholesterol depletion, decreased caveolin, and increased KIT ligand expression, are all independently associated with the activation of Ras/ERK signaling. Upon further analysis, we found that sensitivity to epidermal growth factor activation of ERK phosphorylation was significantly higher, and that cholesterol was significantly depleted, in cells overexpressing HMGA1a. The cumulative evidence indicates that one likely mechanism by which the HMGA1a protein promotes malignant changes in cells is through increased sensitivity to the activation of the Ras/ERK signaling pathway.

Oligonucleotide microarray analysis of gene expression in follicle-stimulating hormone-treated rat Sertoli cells.

Spermatogenesis requires the presence of functional somatic Sertoli cells in the seminiferous tubules of the testis. Sertoli cells provide support and factors necessary for the successful progression of germ cells into spermatozoa. Sertoli cells are regulated to a large degree by the glycoprotein hormone FSH, which is required for the testis to acquire full size and spermatogenic capacity. Signaling events initiated by the binding of FSH to its receptor lead to an alteration of Sertoli cell gene expression. To characterize the changes in gene expression in FSH-treated Sertoli cells, we used the mRNA from these cells to screen Affymetrix U34A rat GeneChip oligonucleotide microarrays. Sertoli cells from 20-d-old rats were cultured in the presence of 25 ng/ml ovine FSH. At 0, 2, 4, 8, and 24 h after the addition of FSH, total RNA was purified and used to prepare biotinylated target, which was hybridized to the U34A rat microarray containing approximately 9000 rat genes. Analysis identified 100-300 transcripts at each time point that were up-regulated or down-regulated by 2-fold or greater. Genes previously reported to be FSH or cAMP regulated in rat Sertoli cells were identified, in addition to numerous genes not reported to be expressed or FSH regulated in Sertoli cells. The expression patterns of five of these genes, encoding nerve growth factor inducible gene B, PRL-1, PC3 nerve growth factor-inducible antiproliferative putative secreted protein, diacylglycerol acyltransferase, and an expressed sequence tag, in FSH- and N,O'-dibutyryl cAMP-treated rat Sertoli cells were confirmed and characterized by Northern blot analysis. Thus, we have begun to define the transcriptome induced and repressed by FSH in rat Sertoli cells, and we have generated datasets of genes available for further analysis in regard to spermatogenesis and Sertoli cell signaling.