Trefoil Factor 3 (TFF3) Is Regulated by Food Intake,Improves Glucose Tolerance and Induces Mucinous Metaplasia

Trefoil factor 3 (TFF3), also called intestinal trefoil factor or Itf, is a 59 amino acid peptide found as a homodimer predominantly along the gastrointestinal tract and in serum. TFF3 expression is elevated during gastrointestinal adenoma progression and has been shown to promote mucosal wound healing. Here we show that in contrast to other trefoil factor family members, TFF1 and TFF2, TFF3 is highly expressed in mouse duodenum, jejunum and ileum and that its expression is regulated by food intake. Overexpression of TFF3 using a recombinant adeno-associated virus (AAV) vector, or daily administration of recombinant TFF3 protein in vivo improved glucose tolerance in a diet-induced obesity mouse model. Body weight, fasting insulin, triglyceride, cholesterol and leptin levels were not affected by TFF3 treatment. Induction of mucinous metaplasia was observed in mice with AAV-mediated TFF3 overexpression, however, no such adverse histological effect was seen after the administration of recombinant TFF3 protein. Altogether these results suggest that the therapeutic potential of targeting TFF3 to treat T2D may be limited.     

Production of adeno-associated viral vectors in insect cells using triple infection: optimization of baculovirus concentration ratios

The production of viral vectors or virus-like particles for gene therapy or vaccinations using the baculovirus expression system is gaining in popularity. Recently, reports of a viral vector based on adeno-associated virus (AAV) produced in insect cells using the baculovirus expression vector system have been published. This system requires the triple infection of cells with baculovirus vectors containing the AAV gene for replication proteins (BacRep), the AAV gene for structural proteins (BacCap), and the AAV vector genome (BacITR). A statistical approach was used to investigate the multiplicities of infection of the three baculoviruses and the results were extended to the production of AAVs containing various transgenes. Highest AAV yields were obtained when BacRep and BacCap, the baculovirus vectors containing genes that code for proteins necessary for the formation of the AAV vector, were added in equal amounts at high multiplicities of infection. These combinations also resulted in the closest ratios of infectious to total AAV particles produced. Overexpression of the AAV structural proteins led to the production of empty AAV capsids, which is believed to overload the cellular machinery, preventing proper encapsidation of the AAV vector transgene, and decreased the viability of the insect cells. Delaying the input of BacCap, to reduce the amount of capsids produced, resulted in lower infectious AAV titers then when all three baculoviruses were put into the system at the same time. The amount of BacITR added to the system can be less than the other two without loss of AAV yield.     

Genotyping of AAV plasmid stocks: quality control in adeno-associated virus vector production

Recombinant adeno-associated virus (rAAV) vectors are a promising tool for gene therapy. When multiple serotypes are handled in the same laboratory during the AAV vector production, it is essential to have means to identify the serotype in a sample and to confirm the absence of cross-contaminating AAV sequences in plasmid stocks as well as end products. Here, we describe the development of a Multiplex AAV Genotyping (MAG) assay to type sensitively and specifically DNA from AAV serotypes 1-12 and to detect AAV2 serotype DNA sequences encoding peptide insertions used to modify tissue tropism. MAG is based on multiplex PCR using type-specific primers and subsequent multiplex hybridization by Luminex. The assay is highly specific, and can easily identify plasmid cross-contaminations. Using 10-fold dilution series, the detection limit was below 10 AAV genomes per PCR. In artificial cross-contamination experiments with a 1,000-fold excess of one AAV serotype versus another one, the contaminating type could be still detected with 10-100 AAV genomes. In a first application, MAG identified successfully cross-contaminated AAV plasmid stocks. In conclusion, MAG is a powerful high-throughput tool in assessing the purity and identity of AAV DNA plasmids and other starting materials used for AAV vector production.     

Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors

Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (serotypes 1, 3, 4, 5, and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, we describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2rep/AAV1cap and AAV2rep/AAV5cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1 x 10(12) to 1 x 10(13)vector genomes/ml.     

基于TCID50检测AAV9载体制品感染性滴度的方法

目的:建立一种基于半数组织培养感染剂量(median tissue culture infective dose,TCID₅₀)检测9型腺相关病毒(adeno-associated virus type 9,AAV9)载体制品感染性滴度的方法。方法:利用含AAV2 rep和cap基因的1型单纯疱疹病毒(herpes simplex virus type1,HSV1)做为辅助病毒与梯度稀释的AAV9载体制品共同感染HEK-293细胞,培养48 h后用实时荧光定量PCR(quantitative real-time PCR,qPCR)扩增AAV特异性反向末端重复序列(inverted terminal repeats,ITR),根据阳性及阴性感染孔数,利用Kärber法计算样品的TCID₅₀。结果:采用携带增强绿色荧光蛋白报告基因的AAV9载体制品确定辅助病毒HSV1-rc最佳感染复数(multiplicity of infection,MOI)为5,AAV9-101的感染性滴度为1.6×10⁹ TCID₅₀/mL。结论:对AAV9载体制品进行感染性滴度检测,且具有可重复性。     

Addition of six-His-tagged peptide to the C terminus of adeno-associated virus VP3 does not affect viral tropism or production

Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6xHis tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6xHis)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6xHis-tagged VP3mutant recombinant AAV. The 6xHis-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6xHis-modified AAV were equivalent to those of wild-type particles. The 6xHis-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6xHis tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6xHis-tagged AAV VP3 capsid protein and to utilize the 6xHis-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.     

The SSV-Seq 2.0 PCR-Free Method Improves the Sequencing of Adeno-Associated Viral Vector Genomes Containing GC-Rich Regions and Homopolymers

Adeno-associated viral vectors (AAV) are efficient engineered tools for delivering genetic material into host cells. The commercialization of AAV-based drugs must be accompanied by the development of appropriate quality control (QC) assays. Given the potential risk of co-transfer of oncogenic or immunogenic sequences with therapeutic vectors, accurate methods to assess the level of residual DNA in AAV vector stocks are particularly important. An assay based on high-throughput sequencing (HTS) to identify and quantify DNA species in recombinant AAV batches is developed. Here, it is shown that PCR amplification of regions that have a local GC content >90% and include successive mononucleotide stretches, such as the CAG promoter, can introduce bias during DNA library preparation, leading to drops in sequencing coverage. To circumvent this problem, SSV-Seq 2.0, a PCR-free protocol for sequencing AAV vector genomes containing such sequences, is developed. The PCR-free protocol improves the evenness of the rAAV genome coverage and consequently leads to a more accurate relative quantification of residual DNA. HTS-based assays provide a more comprehensive assessment of DNA impurities and AAV vector genome integrity than conventional QC tests based on real-time PCR and are useful methods to improve the safety and efficacy of these viral vectors.     

Proteasome inhibition is partially effective in attenuating pre-existing immunity against recombinant adeno-associated viral vectors

Pre-existing immunity against adeno-associated virus (AAV) remains a major challenge facing the clinical use of systemic administration of recombinant AAV vectors for the treatment of genetic and acquired diseases using gene therapy. In this study, we evaluated the potential of bortezomib (marketed under trade name Velcade) to abrogate a pre-existing immunity to AAV in mice, thereby allowing subsequent transduction by a recombinant AAV vector of the same serotype. We demonstrate that bortezomib efficiently reduces AAV-specific IgG titres and moderates the cytotoxic T cell response in mice that have a pre-existing immunity to AAV2/8. Significant depletion of AAV2/8-specific IgG-producing plasma cells in secondary lymphoid organs and bone marrow was observed. However, this inhibition of the immune response by bortezomib was insufficient to allow subsequent re-infection with a recombinant AAV vector of a similar serotype. We show that this shortcoming is probably due to the combination of residual antibody levels and the inability of bortezomib to completely deplete the memory B cells that are re-activated in response to a repeated infection with a recombinant AAV vector. Taken together, the results of this study argue for the use of immunosuppressive therapies that target both plasma and memory B cells for the efficient elimination of pre-existing immunity against AAV2/8 vectors.     

High-titer, wild-type free recombinant adeno-associated virus vector production using intron-containing helper plasmids

Recombinant adeno-associated virus (rAAV) is capable of directing long-term, high-level transgene expression without destructive cell-mediated immune responses. However, traditional packaging methods for rAAV vectors are generally inefficient and contaminated with replication-competent AAV (rcAAV) particles. Although wild-type AAV is not associated with any known human diseases, contaminating rcAAV particles may affect rAAV gene expression and are an uncontrolled variable in many AAV gene transfer studies. In the current study, a novel strategy was designed to both optimize AAV rep gene expression and increase vector yield, as well as simultaneously to diminish the potential of generating rcAAV particles from the helper plasmid. The strategy is based on the insertion of an additional intron in the AAV genome. In the AAV infectious clone, the intron insertion had no effects on the properties of Rep proteins expressed. Normal levels of both Rep and Cap proteins were expressed, and the replication of the AAV genome was not impaired. However, the generation of infectious rcAAV particles using intronized AAV helper was greatly diminished, which was due to the oversized AAV genome caused by the insertion of the artificial introns. Moreover, the rAAV packaging was significantly improved with the appropriate choice of intron and insertion position. The intron is another element that can regulate the rep and cap gene expression from the helper plasmid. This study provides for a novel AAV packaging system which is highly versatile and efficient. It can not only be combined with other AAV packaging systems, including rep-containing cell lines and herpes simplex virus hybrid packaging methods, but also be used in other vector systems as well.     

Enhanced response to enzyme replacement therapy in Pompe disease after the induction of immune tolerance

Pompe disease, which results from mutations in the gene encoding the glycogen-degrading lysosomal enzyme acid alpha -glucosidase (GAA) (also called "acid maltase"), causes death in early childhood related to glycogen accumulation in striated muscle and an accompanying infantile-onset cardiomyopathy. The efficacy of enzyme replacement therapy (ERT) with recombinant human GAA was demonstrated during clinical trials that prolonged subjects' overall survival, prolonged ventilator-free survival, and also improved cardiomyopathy, which led to broad-label approval by the U.S. Food and Drug Administration. Patients who lack any residual GAA expression and are deemed negative for cross-reacting immunologic material (CRIM) have a poor response to ERT. We previously showed that gene therapy with an adeno-associated virus (AAV) vector containing a liver-specific promoter elevated the GAA activity in plasma and prevented anti-GAA antibody formation in immunocompetent GAA-knockout mice for 18 wk, predicting that liver-specific expression of human GAA with the AAV vector would induce immune tolerance and enhance the efficacy of ERT. In this study, a very low number of AAV vector particles was administered before initiation of ERT, to prevent the antibody response in GAA-knockout mice. A robust antibody response was provoked in naive GAA-knockout mice by 6 wk after a challenge with human GAA and Freund's adjuvant; in contrast, administration of the AAV vector before the GAA challenge prevented the antibody response. Most compellingly, the antibody response was prevented by AAV vector administration during the 12 wk of ERT, and the efficacy of ERT was thereby enhanced. Thus, AAV vector-mediated gene therapy induced a tolerance to introduced GAA, and this strategy could enhance the efficacy of ERT in CRIM-negative patients with Pompe disease and in patients with other lysosomal storage diseases.     

Efficient mouse airway transduction following recombination between AAV vectors carrying parts of a larger gene

The small packaging capacity of adeno-associated virus (AAV) vectors limits the utility of this promising vector system for transfer of large genes. We explored the possibility that larger genes could be reconstituted following homologous recombination between AAV vectors carrying overlapping gene fragments. An alkaline phosphatase (AP) gene was split between two such AAV vectors (rec vectors) and packaged using AAV2 or AAV6 capsid proteins. Rec vectors having either capsid protein recombined to express AP in cultured cells at about 1-2% of the rate observed for an intact vector. Surprisingly, the AAV6 rec vectors transduced lung cells in mice almost as efficiently as did an intact vector, with 10% of airway epithelial cells, the target for treatment of cystic fibrosis (CF), being positive. Thus AAV rec vectors may be useful for diseases such as CF that require transfer of large genes.     

Manufacturing of recombinant adeno‐associated viruses using mammalian expression platforms

Manufacturing practices for recombinant adeno‐associated viruses (AAV) have improved in the last decade through the development of new platforms in conjunction with better production and purification methods. In this review, we discuss the advantages and limitations of the most popular systems and methods employed with mammalian cell platforms. Methods and systems such as transient transfection, packaging and producer cells and adenovirus and herpes simplex virus are described. In terms of best production yields, they are comparable with about 10⁴–10⁵ vector genomes produced per cell but transient transfection of HEK293 cells is by far the most commonly used. For small‐scale productions, AAV can be directly purified from the producing cell lysate by ultracentrifugation on a CsCl or iodixanol‐step gradient whereas large‐scale purification requires a combination of multiple steps. Micro/macrofiltration (i.e. including tangential flow filtration and/or dead‐end filtration) and chromatography based‐methods are used for large‐scale purification. Purified AAV products must then be quantified and characterized to ensure quality. Recent purification methods and current analytical techniques are reviewed here. Finally, AAV technology is very promising, but manufacturing improvements are still required to meet the needs of affordable, safe and effective AAV vectors essential for licensing of gene therapy clinical protocols.     

Upscaling of lentiviral vector production by tangential flow filtration

BACKGROUND: HIV-1-derived vectors are promising tools for gene transfer into the brain. Application of these vectors for gene therapy or for the creation of animal models for neurodegenerative diseases requires standardization and upscaling of lentiviral vector production methods. METHODS: In this study, serum-free HIV-1 vector production was efficiently upscaled by use of cell factories and the introduction of tangential flow filtration (TFF) prior to centrifugation. RESULTS: Vector titers (TU/ml) and p24 values (pg p24/ml) for a serum-free HIV-1 vector produced in cell factories and using TFF prior to centrifugation were comparable to those of small-scale productions. TFF allowed a 66-fold concentration of the vectors with complete vector recovery. Further concentration of the vector (30-fold) was achieved either by low-speed centrifugation or by ultracentrifugation. Combination of TFF and ultracentrifugation resulted in a vector recovery of 90-100% and titers that increased 1800-fold and 900-fold for transducing units and p24 concentration, respectively. CONCLUSIONS: With this new standardized method for lentiviral vector production and concentration, 1 ml of concentrated vector is routinely produced with titers of 10(9)-10(10) TU/ml starting from 2 l of cell-culture medium. Moreover, stereotactic injection of this vector in mouse striatum resulted in a large transduced brain volume in the absence of any immune response.     

Characterization of tissue tropism determinants of adeno-associated virus type 1

Muscle is an attractive target for gene delivery because of its mass and because vectors can be delivered in a noninvasive fashion. Adeno-associated virus (AAV) has been shown to be effective for muscle-targeted gene transfer. Recent progress in characterization of AAV serotype 1 (AAV1) and AAV6 demonstrated that these two AAV serotypes are far more efficient in transducing muscle than is the traditionally used AAV2. Since all cis elements are identical in these vectors, the potential determinants for their differences in transducing muscle appear to be located within the AAV capsid proteins. In the present study, a series of AAV capsid mutants were generated to identify the major regions affecting AAV transduction efficiency in muscle. Replacement of amino acids 350 to 736 of AAV2 VP1 with the corresponding amino acids from VP1 of AAV1 resulted in a hybrid vector that behaved very similarly to AAV1 in vitro and in vivo in muscle. Characterization of additional mutants carrying smaller regions of the AAV1 VP1 amino acid sequence in the AAV2 capsid protein suggested that amino acids 350 to 430 of VP1 function as a major tissue tropism determinant. Further analysis showed that the heparin binding domain and the major antigenic determinants in the AAV capsid region were not necessary for the efficiency of AAV1 transduction of muscle.     

Production and titering of recombinant adeno-associated viral vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.     

High performance anion exchange chromatography purification of probiotic bacterial extracellular vesicles enhances purity and anti-inflammatory efficacy

Bacterial extracellular vesicles (BEVs), including outer membrane vesicles, have emerged as a promising new class of vaccines and therapeutics to treat cancer and inflammatory diseases, among other applications. However, clinical translation of BEVs is hindered by a current lack of scalable and efficient purification methods. Here, we address downstream BEV biomanufacturing limitations by developing a method for orthogonal size- and charge-based BEV enrichment using tangential flow filtration (TFF) in tandem with high performance anion exchange chromatography (HPAEC). The data show that size-based separation coisolated protein contaminants, whereas size-based TFF with charged-based HPAEC dramatically improved purity of BEVs produced by probiotic Gram-negative Escherichia coli and Gram-positive lactic acid bacteria (LAB). Escherichia coli BEV purity was quantified using established biochemical markers while improved LAB BEV purity was assessed via observed potentiation of anti-inflammatory bioactivity. Overall, this work establishes orthogonal TFF + HPAEC as a scalable and efficient method for BEV purification that holds promise for future large-scale biomanufacturing of therapeutic BEV products.     

A novel terminal resolution-like site in the adeno-associated virus type 2 genome.

The adeno-associated virus 2 (AAV) contains a single-stranded DNA genome of which the terminal 145 nucleotides are palindromic and form T-shaped hairpin structures. These inverted terminal repeats (ITRs) play an important role in AAV DNA replication and resolution, since each of the ITRs contains a terminal resolution site (trs) that is the target site for the AAV rep gene products (Rep). However, the Rep proteins also interact with the AAV DNA sequences that lie outside the ITRs, and the ITRs also play a crucial role in excision of the proviral genome from latently infected cells or from recombinant AAV plasmids. To distinguish between Rep-mediated excision of the viral genome during rescue from recombinant AAV plasmids and the Rep-mediated resolution of the ITRs during AAV DNA replication, we constructed recombinant AAV genomes that lacked either the left or the right ITR sequence and one of the Rep-binding sites (RBSs). No rescue and replication of the AAV genome occurred from these plasmids following transfection into adenovirus type 2-infected human KB cells, as expected. However, excision and abundant replication of the vector sequences was clearly detected from the plasmid that lacked the AAV left ITR, suggesting the existence of an additional putative excision site in the left end of the AAV genome. This site was precisely mapped to one of the AAV promoters at map unit 5 (AAV p5) that also contains an RBS. Furthermore, deletion of this RBS abolished the rescue and replication of the vector sequences. These studies suggest that the Rep-mediated cleavage at the RBS during viral DNA replication may, in part, account for the generation of the AAV defective interfering particles.     

High performance tangential flow filtration

Conventional tangential flow filtration (TFF) has traditionally been limited to separation of solutes that differ by about ten-fold in size. Wide pore-size distributions, membrane fouling, and concentration polarization phenomena have commonly been cited as reasons for this limitation. The use of TFF in the biotechnology industry has therefore been restricted to cell-protein, virus-protein, and protein-buffer separations. A multi-disciplinary team with industrial and academic members was formed to overcome these limitations and enable protein-protein separations using High Performance TFF (HPTFF) systems. Pore-size distributions have been improved with the development of new membrane formulation and casting techniques. Membrane fouling has been controlled by operating in the transmembrane pressure-dependent regime of the filtrate flux curve and by carefully controlling fluid dynamic start-up conditions. Concentration polarization was exploited to enhance, rather than limit, the resolution of solutes. Concentration polarization has also been controlled by operating a co-current filtrate stream that maintains transmembrane pressure constant along the length of the TFF module. High yields and purification factors were obtained even with small differences in protein sieving. IgG-BSA and BSA monomer-oligomer mixtures have successfully been separated with these systems. HPTFF technology provides a competitive purification tool to complement chromatographic processing of proteins. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 71-82, 1997.     

Optimized production of high-titer recombinant adeno-associated virus in roller bottles

Adeno-associated viral (AAV) vectors are used for in vivo gene transfer in a number of preclinical models of genetic diseases (including large-animal models) and are currently being tested in clinical trials for treatment of hemophilia B and cystic fibrosis. Protocols for production of AAV vectors in a helper virus-free system are available and are based on transient transfection of HEK-293 cells with multiple plasmids. Scale-up of vector production has been labor intensive and inefficient because of a lack of larger culture vessels suitable for growth of adherent cells, large-scale transfection, and vector production. Here we report efficient production of AAV vector in roller bottles, which represents a 10-fold scale-up from the conventional flask or plate method. Optimized production yielded greater than 10(13) vector genomes per bottle and was as cost effective as published protocols using plates. Successful vector production by this method was dependent on optimization of transfection by calcium phosphate precipitation, of monitoring of cell growth (by measurement of glucose consumption), of cell culture conditions, and CO2/air exchange with the culture vessel.