Micro-dystrophin AAV Vectors Made by Transient Transfection and Herpesvirus System Are Equally Potent in Treating mdx Mouse Muscle Disease.

Vector production scale-up is a major barrier in systemic adeno-associated virus (AAV) gene therapy. Many scalable manufacturing methods have been developed. However, the potency of the vectors generated by these methods has rarely been compared with vectors made by transient transfection (TT), the most commonly used method in preclinical studies. In this study, we blindly compared therapeutic efficacy of an AAV9 micro-dystrophin vector generated by the TT method and scalable herpes simplex virus (HSV) system in a Duchenne muscular dystrophy mouse model. AAV was injected intravenously at 5 × 1014 (high), 5 × 1013 (medium), or 5 × 1012 (low) viral genomes (vg)/kg. Comparable levels of micro-dystrophin expression were observed at each dose in a dose-dependent manner irrespective of the manufacturing method. Vector biodistribution was similar in mice injected with either the TT or the HSV method AAV. Evaluation of muscle degeneration/regeneration showed equivalent protection by vectors made by either method in a dose-dependent manner. Muscle function was similarly improved in a dose-dependent manner irrespective of the vector production method. No apparent toxicity was observed in any mouse. Collectively, our results suggest that the biological potency of the AAV micro-dystrophin vector made by the scalable HSV method is comparable to that made by the TT method.

Molecular characterization of precise in vivo targeted gene integration in human cells using AAVHSC15.

Targeted gene integration via precise homologous recombination (HR)-based gene editing has the potential to correct genetic diseases. AAV (adeno-associated virus) can mediate nuclease-free gene integration at a disease-causing locus. Therapeutic application of AAV gene integration requires quantitative molecular characterization of the edited sequence that overcome technical obstacles such as excess episomal vector genomes and lengthy homology arms. Here we describe a novel molecular methodology that utilizes quantitative next-generation sequencing to characterize AAV-mediated targeted insertion and detects the presence of unintended mutations. The methods described here quantify targeted insertion and query the entirety of the target locus for the presence of insertions, deletions, single nucleotide variants (SNVs) and integration of viral components such as inverted terminal repeats (ITR). Using a humanized liver murine model, we demonstrate that hematopoietic stem-cell derived AAVHSC15 mediates in vivo targeted gene integration into human chromosome 12 at the PAH (phenylalanine hydroxylase) locus at 6% frequency, with no sign of co-incident random mutations at or above a lower limit of detection of 0.5% and no ITR sequences at the integration sites. Furthermore, analysis of heterozygous variants across the targeted locus using the methods described shows a pattern of strand cross-over, supportive of an HR mechanism of gene integration with similar efficiencies across two different haplotypes. Rapid advances in the application of AAV-mediated nuclease-free target integration, or gene editing, as a new therapeutic modality requires precise understanding of the efficiency and the nature of the changes being introduced to the target genome at the molecular level. This work provides a framework to be applied to homologous recombination gene editing platforms for assessment of introduced and natural sequence variation across a target site.

Sustained Correction of a Murine Model of Phenylketonuria following a Single Intravenous Administration of AAVHSC15-PAH.

Phenylketonuria is an inborn error of metabolism caused by loss of function of the liver-expressed enzyme phenylalanine hydroxylase and is characterized by elevated systemic phenylalanine levels that are neurotoxic. Current therapies do not address the underlying genetic disease or restore the natural metabolic pathway resulting in the conversion of phenylalanine to tyrosine. A family of hepatotropic clade F adeno-associated viruses (AAVs) was isolated from human CD34+ hematopoietic stem cells (HSCs) and one (AAVHSC15) was utilized to deliver a vector to correct the phenylketonuria phenotype in Pahenu2 mice. The AAVHSC15 vector containing a codon-optimized form of the human phenylalanine hydroxylase cDNA was administered as a single intravenous dose to Pahenu2 mice maintained on a phenylalanine-containing normal chow diet. Optimization of the transgene resulted in a vector that produced a sustained reduction in serum phenylalanine and normalized tyrosine levels for the lifespan of Pahenu2 mice. Brain levels of phenylalanine and the downstream serotonin metabolite 5-hydroxyindoleacetic acid were restored. In addition, the coat color of treated mice darkened following treatment, indicating restoration of the phenylalanine metabolic pathway. Taken together, these data support the potential of an AAVHSC15-based gene therapy as an investigational therapeutic for phenylketonuria patients.

Improved cell-specificity of adeno-associated viral vectors for medullary thyroid carcinoma using calcitonin gene regulatory elements.

Targeted gene therapy using recombinant adeno-associated virus (rAAV) vectors is a potential therapeutic strategy for treating cancer, and tissue-specific promoters may help with tissue targeting. Medullary thyroid carcinoma (MTC) is a disease of the calcitonin secreting thyroid C cells, and calcitonin is highly expressed in MTC tumors compared to other cells. To target MTC cells, we evaluated an rAAV serotype 2 vector (rAAV2-pM+104-GFP) containing a modified calcitonin/calcitonin gene related peptide promoter (pM+104) and a green fluorescent protein (GFP) reporter gene. In vitro transduction experiments comparing the MTC TT cell line with non-MTC cell lines demonstrated that rAAV2-pM+104-GFP infection yielded significantly (p < 0.05) higher GFP expression in TT cells than in non-MTC cell lines (HEK293 and HeLa), and significantly higher expression than in TT cells infected with the positive control rAAV2-pCBA-GFP vector. The rAAV2-pCBA-GFP control vector included a well-characterized, ubiquitously expresses control promoter, the chicken beta actin promoter with a cytomegalovirus enhancer (pCBA). In vivo experiments using a TT cell xenograft tumor mouse model showed that tumors directly injected with 2 x 1010 vg of rAAV2-pM+104-GFP vector resulted in GFP expression detected in 21.7% of cells, 48 hours after the injection. Furthermore, GFP expression was significantly higher for rAAV-pM+104-GFP treatments with a longer vector treatment duration and higher vector dose, with up to 52.6% (q < 0.05) GFP cells detected 72 hours after injecting 1x 1011 vg/tumor. These data show that we have developed an rAAV vector with improved selectivity for MTC.

Sodium Chloride Enhances Recombinant Adeno-Associated Virus Production in a Serum-Free Suspension Manufacturing Platform Using the Herpes Simplex Virus System.

The increase in effective treatments using recombinant adeno-associated viral (rAAV) vectors has underscored the importance of scalable, high-yield manufacturing methods. Previous work from this group reported the use of recombinant herpes simplex virus type 1 (rHSV) vectors to produce rAAV in adherent HEK293 cells, demonstrating the capacity of this system and quality of the product generated. Here we report production and optimization of rAAV using the rHSV system in suspension HEK293 cells (Expi293F) grown in serum and animal component-free medium. Through adjustment of salt concentration in the medium and optimization of infection conditions, titers greater than 1 × 1014 vector genomes per liter (VG/liter) were observed in purified rAAV stocks produced in Expi293F cells. Furthermore, this system allowed for high-titer production of multiple rAAV serotypes (2, 5, and 9) as well as multiple transgenes (green fluorescent protein and acid α-glucosidase). A proportional increase in vector production was observed as this method was scaled, with a final 3-liter shaker flask production yielding an excess of 1 × 1015 VG in crude cell harvests and an average of 3.5 × 1014 total VG of purified rAAV9 material, resulting in greater than 1 × 105 VG/cell. These results support the use of this rHSV-based rAAV production method for large-scale preclinical and clinical vector production.

A scalable method for the production of high-titer and high-quality adeno-associated type 9 vectors using the HSV platform.

Recombinant adeno-associated vectors based on serotype 9 (rAAV9) have demonstrated highly effective gene transfer in multiple animal models of muscular dystrophies and other neurological indications. Current limitations in vector production and purification have hampered widespread implementation of clinical candidate vectors, particularly when systemic administration is considered. In this study, we describe a complete herpes simplex virus (HSV)-based production and purification process capable of generating greater than 1 × 10(14) rAAV9 vector genomes per 10-layer CellSTACK of HEK 293 producer cells, or greater than 1 × 10(5) vector genome per cell, in a final, fully purified product. This represents a 5- to 10-fold increase over transfection-based methods. In addition, rAAV vectors produced by this method demonstrated improved biological characteristics when compared to transfection-based production, including increased infectivity as shown by higher transducing unit-to-vector genome ratios and decreased total capsid protein amounts, shown by lower empty-to-full ratios. Together, this data establishes a significant improvement in both rAAV9 yields and vector quality. Further, the method can be readily adapted to large-scale good laboratory practice (GLP) and good manufacturing practice (GMP) production of rAAV9 vectors to enable preclinical and clinical studies and provide a platform to build on toward late-phases and commercial production.

Parvovirus and thyroid cancer.

Parvoviruses are some of the smallest DNA viruses known to infect a wide range of animal species and humans. Though not all parvoviruses are pathogenic, some can cause disease ranging from asymptomatic to benign to life-threatening. Recently, there has been an interest in the possible role of parvoviruses in thyroid disease in general. The objectives of this review are to cite and appraise the available evidence on the role of parvoviruses in thyroid cancer in particular. Little to no evidence is available directly linking animal parvoviruses and thyroid cancer, but there is a growing literature on the human erythrovirus B19 (EVB19) and its association with thyroid cancer. Of particular interest is the persistence and expression of EVB19 DNA, RNA, and protein in a wide variety of thyroid tissues. While a causative role of EVB19 in the pathogenesis of thyroid cancer cannot be supported at this time, an indirect role is hypothesized and discussed but with the recognition that the data are limited. Further studies are clearly warranted to determine the exact, if any, role of this human pathogen in thyroid cancer.

Persistent parvovirus B19 infection in non-erythroid tissues: possible role in the inflammatory and disease process.

Parvovirus B19 (B19V) is a small non-enveloped DNA virus of the Parvoviridae family. It is an obligate human pathogen that preferentially replicates in erythroid progenitor cells. B19V is the causative agent of multiple erythroid-related diseases due to replication-induced cytotoxicity. Despite its strong erythroid tropism and related acute disease association, B19V has been determined to persist in many other non-erythroid tissues. This review summarizes and appraises what is known about concomitant B19V DNA persistence and non-acute viral gene expression in various, particularly non-erythroid, tissue types. The methods utilized for B19V detection are described, focusing on the discrepancies in outcomes among the employed assays. The studies where investigations focused on the impact of persistent B19V expression on cellular signaling pathways are also summarized. These studies demonstrate the expanse of the types of cells capable of in vivo B19V expression as well as the possible effect of persistent viral infection on the cellular microenvironment. Overall, these reports indicate that B19V commonly persists in a wide range of both erythroid and non-erythroid tissues, and that low-level viral gene expression can be detected in some persistently infected cells. B19V capsid RNA or proteins have been reported in bone marrow, colon, heart, liver, lymphoid, synovial, testicular, and thyroid tissues. In a sub-set of these cases, B19V capsid mRNA or proteins have been associated with increased inflammatory-related gene expression. The development of standard protocols to assay for B19V infection and expression in the context of non-erythroid, non-acute disease is warranted, and with further targeted studies, may begin to elucidate the impact of persistent B19V infection in vivo. These studies may determine the most conducive cellular environment for persistent gene expression and possible impact on disease pathogenesis.

Parvovirus b19 persistence in abnormal thyroid tissue of a mature cystic ovarian teratoma: a case report.

Ovarian teratomas represent the most common neoplasm derived from germ cells and can contain mature ectodermal, mesodermal, and endodermal tissues. In rare cases, these teratomas can be composed predominantly or solely of thyroid tissue. These thyroid cells often function similarly to normal thyroid tissues. This laboratory and others have previously shown that parvovirus B19 (B19V) persists in primary and metastatic thyroid tissues. No reports exist on possible B19V persistence in thyroid tissues that may arise de novo outside the thyroid gland proper. In this case report, the detection of B19V (genotype 1) in the thyroid epithelial cells of a mature teratoma is reported. Nested PCR and immunohistochemistry were used to detect viral nucleic acids and proteins, respectively. Viral genomes were amplified in lesion DNA, confirming persistence of B19V. Positive immunohistochemical staining was seen for B19V capsid proteins in the thyroid epithelial cells within the mature teratoma, but not in surrounding ovarian tissue or in the non-thyroidal elements of the mature teratoma. These results demonstrate for the first time that thyroid epithelial cells, derived from non-thyroid tissue, are capable of supporting B19V infection and persistence.