Mechanistic modeling explains the production dynamics of recombinant adeno-associated virus with the baculovirus expression vector system.

Current manufacturing processes for recombinant adeno-associated viruses (rAAVs) have less-than-desired yields and produce significant amounts of empty capsids. The increasing demand and the high cost of goods for rAAV-based gene therapies motivate development of more efficient manufacturing processes. Recently, the US Food and Drug Administration (FDA) approved the first rAAV-based gene therapy product manufactured in the baculovirus expression vector system (BEVS), a technology that demonstrated production of high titers of full capsids. This work presents a first mechanistic model describing the key extracellular and intracellular phenomena occurring during baculovirus infection and rAAV maturation in the BEVS. The model predictions are successfully validated for in-house and literature experimental measurements of the vector genome and of structural and non-structural proteins collected during rAAV manufacturing in the BEVS with the TwoBac and ThreeBac constructs. A model-based analysis of the process is carried out to identify the bottlenecks that limit full capsid formation. Vector genome amplification is found to be the limiting step for rAAV production in Sf9 cells using either the TwoBac or ThreeBac system. In turn, vector genome amplification is hindered by limiting Rep78 levels. Transgene and non-essential baculovirus protein expression in the insect cell during rAAV manufacturing also negatively influences the rAAV production yields.

Germline viral "fossils" guide in silico reconstruction of a mid-Cenozoic era marsupial adeno-associated virus.

Germline endogenous viral elements (EVEs) genetically preserve viral nucleotide sequences useful to the study of viral evolution, gene mutation, and the phylogenetic relationships among host organisms. Here, we describe a lineage-specific, adeno-associated virus (AAV)-derived endogenous viral element (mAAV-EVE1) found within the germline of numerous closely related marsupial species. Molecular screening of a marsupial DNA panel indicated that mAAV-EVE1 occurs specifically within the marsupial suborder Macropodiformes (present-day kangaroos, wallabies, and related macropodoids), to the exclusion of other Diprotodontian lineages. Orthologous mAAV-EVE1 locus sequences from sixteen macropodoid species, representing a speciation history spanning an estimated 30 million years, facilitated compilation of an inferred ancestral sequence that recapitulates the genome of an ancient marsupial AAV that circulated among Australian metatherian fauna sometime during the late Eocene to early Oligocene. In silico gene reconstruction and molecular modelling indicate remarkable conservation of viral structure over a geologic timescale. Characterisation of AAV-EVE loci among disparate species affords insight into AAV evolution and, in the case of macropodoid species, may offer an additional genetic basis for assignment of phylogenetic relationships among the Macropodoidea. From an applied perspective, the identified AAV "fossils" provide novel capsid sequences for use in translational research and clinical applications.

Long-term restoration of cardiac dystrophin expression in golden retriever muscular dystrophy following rAAV6-mediated exon skipping.

Although restoration of dystrophin expression via exon skipping in both cardiac and skeletal muscle has been successfully demonstrated in the mdx mouse, restoration of cardiac dystrophin expression in large animal models of Duchenne muscular dystrophy (DMD) has proven to be a challenge. In large animals, investigators have focused on using intravenous injection of antisense oligonucleotides (AO) to mediate exon skipping. In this study, we sought to optimize restoration of cardiac dystrophin expression in the golden retriever muscular dystrophy (GRMD) model using percutaneous transendocardial delivery of recombinant AAV6 (rAAV6) to deliver a modified U7 small nuclear RNA (snRNA) carrying antisense sequence to target the exon splicing enhancers of exons 6 and 8 and correct the disrupted reading frame. We demonstrate restoration of cardiac dystrophin expression at 13 months confirmed by reverse transcription-PCR (RT-PCR) and immunoblot as well as membrane localization by immunohistochemistry. This was accompanied by improved cardiac function as assessed by cardiac magnetic resonance imaging (MRI). Percutaneous transendocardial delivery of rAAV6 expressing a modified U7 exon skipping construct is a safe, effective method for restoration of dystrophin expression and improvement of cardiac function in the GRMD canine and may be easily translatable to human DMD patients.

Reproducible high yields of recombinant adeno-associated virus produced using invertebrate cells in 0.02- to 200-liter cultures.

The large amounts of recombinant adeno-associated virus (rAAV) vector needed for clinical trials and eventual commercialization require robust, economical, reproducible, and scalable production processes compatible with current good manufacturing practice. rAAV produced using baculovirus and insect cells satisfies these conditions; however, recovering rAAV particles from 200-liter bioreactors is more complicated than bench-scale vector preparations. Using a variety of processing media, we developed a reliable and routine downstream procedure for rAAV production that is scalable from 0.02- to 200-liter cultures. To facilitate the upstream process, we adapted the titerless infected-cell preservation and scale-up process for rAAV production. Single-use aliquots of cryopreserved baculovirus-infected insect cells (BIIC) are thawed and added to the suspension culture to achieve the desired ratio of BIIC to rAAV-producer cells. By using conditions established with small-scale cultures, rAAV was produced in larger volume cultures. Strikingly consistent rAAV yields were attained in cultures ranging from 10 liters to 200 liters. Based on the final yield, each cell produced 18,000 ± 6,800 particles of purified rAAV in 10-, 20-, 100-, and 200-liter cultures. Thus, with an average cell density of 4.32 × 10(6) cells/ml, ≥ 10(16) purified rAAV particles are produced from 100 to 200 liters. The downstream process resulted in about 20% recovery estimated from comparing the quantities of capsid protein antigen in the crude bioreactor material and in the final, purified product. The ease and reproducibility of rAAV production in 200-liter bioreactors suggest that the limit has not been reached, and 500-liter productions are planned.

High-affinity alphavbeta3 integrin targeted optical probe as a new imaging biomarker for early atherosclerosis: initial studies in Watanabe rabbits.

PURPOSE: A newly developed synthetic alpha v beta 3 integrin targeted optical probe (ITOP) has been demonstrated to target cancer cells, in vivo. Compared to the commercially available cyclic peptide c[RGDfv], this optical probe has at least 20 times better binding affinity for the alpha v beta 3 receptor. The present in vitro study was designed to investigate the possibility of detecting early atherosclerotic plaque by using this ITOP. PROCEDURES: Experiments were performed on five Watanabe heritable hyperlipidemic rabbits and two New Zealand White rabbits for control. Our ITOP was used for detecting the presence of alpha v beta 3 receptors in vitro. RESULTS: Segments of plaque accumulation from two distinct regions of ascending and descending aortas were labeled in Watanabe rabbits. The signal was found principally in the adventitia and proximal intima of the aortic vessel, corresponding directly to the expression of integrin alpha v beta 3 as determined by antibody assay. Moreover, there was a close association between the level of labeling with the alpha v beta 3 targeted probe and the thickness of the adventitia. CONCLUSIONS: This high-affinity ITOP identifies the site and extent of alpha v beta 3 expression and correlates with adventitial thickness. Recent evidence associates alpha v beta 3 expression with the inflammatory process in early vulnerable plaque, making this compound a promising potential biomarker for early atherosclerotic disease.

Producing recombinant adeno-associated virus in foster cells: overcoming production limitations using a baculovirus-insect cell expression strategy.

Establishing pharmacological parameters, such as efficacy, routes of administration, and toxicity, for recombinant adeno-associated virus (rAAV) vectors is a prerequisite for gaining acceptance for clinical applications. In fact, even a therapeutic window, that is, the dose range between therapeutic efficacy and toxicity, has yet to be determined for rAAV in vivo. Multiphase clinical trials investigating the safety and efficacy of recombinant AAV-based therapeutics will require unprecedented vector production capacity to meet the needs of preclinical toxicology studies, and the progressive clinical protocol phases of safety/dose escalation (phase I), efficacy (phase II), and high-enrollment, multicenter evaluations (phase III). Methods of rAAV production capable of supporting such trials must be scalable, robust, and efficient. We have taken advantage of the ease of scalability of nonadherent cell culture techniques coupled with the inherent efficiency of viral infection to develop an rAAV production method based on recombinant baculovirus-mediated expression of AAV components in insect-derived suspension cells.

Reaching for the other side: generating sequence-dependent interstrand cross-links with 5-bromodeoxyuridine and gamma-rays.

Interstrand cross-links impede critical cellular processes such as transcription and replication and are thus considered to be one of the most toxic types of DNA damage. Although several studies now point to the existence of gamma-radiation-induced cross-links in cellular DNA, little is known about the characteristics required for their creation. Recently, we reported the formation of interstrand cross-links that were specific for mismatched nucleotides within 5-bromo-2'-deoxyuridine-substituted DNA. Given the structural specificity for interstrand cross-link formation, it is likely that open or mismatched regions of DNA in cells may be particularly favorable for cross-link production. Herein, we investigated the effect of the local DNA sequence on the formation of interstrand cross-links, using 5-bromo-2'-deoxyuridine to generate radicals in a mismatched region of DNA. We investigated a total of 12 variations of bases in the mismatched region. The oligonucleotides were irradiated with gamma-rays, and interstrand cross-link formation was analyzed by denaturing gel electrophoresis. We found that the efficiency of cross-link formation was highly dependent on the nature of mismatched bases and, on the basis of electrophoretic mobility, observed several distinctive cross-link structures with specific DNA sequences. This study provides new insights into the reactivity of mismatched DNA and the mechanisms leading to interstrand cross-link formation. The potential application of 5-bromo-2'-deoxyuridine-induced interstrand cross-links to the field of DNA repair is discussed.

Evidence of prior exposure to human bocavirus as determined by a retrospective serological study of 404 serum samples from adults in the United States.

Recently, molecular screening for pathogenic agents has identified a partial genome of a novel parvovirus, called human bocavirus (HBoV). The presence of this newly described parvovirus correlated with upper and lower respiratory tract infections in children. Lower respiratory tract infections are a leading cause of hospital admission in children, and the etiological agent has not been identified in up to 39% of these cases. Using baculovirus expression vectors (BEVs) and an insect cell system, we produced virus-like particles (VLPs) of HBoV. The engineered BEVs express the HBoV capsid proteins stoichiometrically from a single open reading frame. Three capsid proteins assemble into the VLP rather than two proteins predicted from the HBoV genome sequence. The denatured capsid proteins VP1, VP2, and VP3 resolve on silver-stained sodium dodecyl sulfate-polyacrylamide gels as three bands with apparent molecular masses of 72 kDa, 68 kDa, and 62 kDa, respectively. VP2 apparently initiates at a GCT codon (alanine) 273 nucleotides downstream from the VP1 start site and 114 nucleotides upstream from the VP3 initiation site. We characterized the stable capsids using physical, biochemical, and serological techniques. We found that the density of the VLP is 1.32 g/cm(3) and is consistent with an icosahedral symmetry with approximately a 25-nm diameter. Rabbit antiserum against the capsid of HBoV, which did not cross-react with adeno-associated virus type 2, was used to develop enzyme-linked immunosorbent assays (ELISAs) for anti-HBoV antibodies in human serum. Using ELISA, we tested 404 human serum samples and established a range of antibody titers in a large U.S. adult population sample.

Interstrand cross-link induction by UV radiation in bromodeoxyuridine-substituted DNA: dependence on DNA conformation.

DNA interstrand cross-links (ICL) can be induced both by natural products (e.g., psoralens with UVA) and by chemical agents, some of which are used in chemotherapy (e.g., Carboplatin and mitomycin C). Here, we report the formation of ICL by UV radiation in brominated DNA, but only for very specific conformations. The quantum yields for strand break and cross-link formation depend on the wavelength with a maximum near 280 nm. It is known that the photosensitization of DNA by bromodeoxyuridine (BrdUrd) results mainly from the electron affinity of bromine, leading to the irreversible formation of 2'-deoxyuridin-5-yl radicals (dUrd*) upon the addition of an electron from an adjacent adenosine. It is well documented that the photolytic loss of the bromine atom is greatly suppressed in single-stranded DNA versus that in double-stranded DNA. To study this behavior, we have used two models of BrdUrd-mediated sensitization: one consists of a DNA duplex containing a bulge, formed by five mismatched bases, including the BrdUrd, and the other consists of completely duplex DNA. UV irradiation induces much higher levels of single-strand breaks (ssb) in the completely duplex DNA at the BrdUrd site compared to the DNA with a bulge. However, in completely duplex DNA, ssb appear only in the brominated strand, whereas in the bulged duplex DNA, ssb occur on both strands. Most importantly, we also observe formation of interstrand cross-links in bulged duplex DNA in the BrdUrd region. Thus, we propose that UV irradiation of cells containing BrdUrd incorporated randomly into duplex DNA will create many ssb, whereas BrdUrd present in DNA bulges or open regions in double-stranded DNA (transcription bubbles, replication forks) will lead to potentially lethal damage in both strands in the form of ICL. These findings may help explain the potent clinical antiviral activity of IdUrd and BrdUrd (e.g., IdUrd is used to treat eye infections caused by the herpes virus) and suggest that ICL formation may be a very specific probe for identifying single-stranded regions in the DNA of living cells. In addition, this model system provides an excellent means of introducing ICL for studies on their repair and biological consequences.

Interstrand cross-links: a new type of gamma-ray damage in bromodeoxyuridine-substituted DNA.

Interstrand cross-links (ICL) represent one of the most toxic types of DNA damage for dividing cells. They are induced both by natural products (e.g., psoralens + UVA) and by several chemical agents, some of which are used in chemotherapy (e.g., carboplatin and mitomycin C). Although repair mechanisms exist for interstrand cross-links, these lesions can induce mutations, chromosomal rearrangements, and cell death. Here, we report, for the first time, the formation of ICL by gamma-rays in brominated DNA. It is well established that the radiosensitization properties of bromodeoxyuridine (BrdUrd) result primarily from the electrophilic nature of the bromine, making it a good leaving group and leading to the irreversible formation of a uridinyl radical (dUrd(*)) or uridinyl anion (dUrd-) upon addition of an electron. We observe that the radiolytic loss of the bromine atom is greatly suppressed in double-stranded compared to single-stranded DNA. We have used a model DNA containing a bulge, formed by five mismatched bases, and have observed a linear dose-response for the formation of strand breaks on the single-stranded regions of both the brominated strand and the opposite nonbrominated strand. Surprisingly, we have observed the formation of interstrand cross-links exclusively in the mismatched region. Thus, we propose that the radiosensitization effects of bromodeoxyuridine in vivo will almost certainly be limited to single strand regions such as found in transcription bubbles, replication forks, mismatched DNA, and possibly the loop region of telomeres. Our results suggest that interstrand cross-links may contribute to the radiosensitization effects of BrdUrd. These findings may have profound implications for the clinical use of bromodeoxyuridine as a radiosensitizer, as well as for the development of targeted radiosensitizers.

Dynamic conformational states of DNA containing T.T or BrdU.T mispaired bases: wobble H-bond pairing versus cross-strand inter-atomic contacts.

The dynamic structure of 11-mer DNA duplexes of different sequences with or without homopyrimidine (T.T, or BrdU.T) mismatches was studied by molecular dynamics (MD) simulations on a time scale from 200 ps to 1 ns. The conformational analysis suggests that in mismatched duplexes the formation of classical T.T wobble H-bonding pairing is nearest-neighbor sequence-dependent and, in most cases, three-centered H-bonds and numerous alternative close cross-strand interatomic contacts exist. Thus, in duplex W1, where the central triplet is 5'd(CTA).d(TTG), two wobble conformations W upward arrow (alphabeta) and W downward arrow (betaalpha) are formed and exchange rapidly at 300 K. In contrast, when the central triplet is 5'd(TTT).d(ATA) (W2 duplex) wobble conformations are rarely observed at 300 K, and the T.T mispair most often adopts a "twisted" conformation with one largely persistent normal H-bond, plus a stable cross-strand contact involving a T flanking base. However, at elevated temperature (400 K) the same W2 duplex shows frequent exchange between the two classical wobble conformations (alphabeta<-->betaalpha), as is in the case when the central triplet is 5'd(TBrdUT).d(ATA) (W3 duplex at 300 K). It is suggested that in the W2 sequence, restrictions due to thymine-methyl/pi interactions prevent the formation of wobble pairing and thermal activation energy, and/or the chemical replacement of T by BrdU are required in order for the T(BrdU).T mismatch to adopt and exchange between wobble conformations. The specific short and/or long-lived (double/triple) cross-strand dynamic interactions in W1, W2 and W3 duplexes are throughout characterized. These frequent atomic encounters exemplify possible inter-strand charge transfer pathways in the studied DNA molecules.