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Order Piccovirales
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
Furthermore, the random peptide libraries were displayed on the AAV2 vector to select for targeted gene therapy vectors on specific cells (Müller et al. 2003; Waterkamp et al. 2006; Naumer et al. 2012a, b). Adachi and Nakai (2010) engineered a novel peptide display library platform based on the AAV1 virion with aa 445–568 being replaced with those of AAV9 in order to impair infectivity and delay blood clearance. The insect cells-produced AAV2 VLPs were coated with polyethyleneimine to ensure efficient siRNA delivery in breast cancer therapy (Shao et al. 2012). Lee NC et al. (2012) improved axial muscle tropism and decreased liver tropism by insertion of acidic six aspartic acids D6 oligopeptide into heparan sulfate proteoglycan binding region within the AAV2 VP1. Kienle et al. (2012) generated the synthetic AAV vectors by DNA shuffling. The targeting to the cancer cell-surface marker EGFR was improved by fusion of two modular targeting molecules (DARPin or Affibody) to N-terminus of the AAV2 VP2 (Hagen et al. 2014). Pandya et al. (2014) ensured the specific targeting to dendritic cells by mutational modification of the surface of the AAV6 vector. The visualization of the AAV vectors was achieved by replacement of VP2 by the VP2 with N-terminally fused eGFP protein (Lux et al. 2005).
Biocatalysts: The Different Classes and Applications for Synthesis of APIs
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Xu et al. (2017) described an alcohol dehydrogenase discovered from Klebsiella oxytoca (KleADH)by a genome mining method, which was able to convert t-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate to t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate, the key intermediate for producing statins such as rosuvastatin and atorvastatin (see also below). The enzyme was overexpressed in Escherichia coli Rosetta (DE3) and the use of the whole cell biocatalyst made an addition of expensive cofactors redundant. Ma et al. (2010) employed a ketoreductase (KRED) for the enantioselective reduction of ethyl-4-chloroaceto acetate combined with the GDH/glucose system for NADP regeneration; the product, ethyl (S)-4-chloro-3-hydroxybutyrate was then subjected to a treatment with a halohydrin dehalogenase (HHDH) in presence of NaCN to substitute a cyano residue by the Cl moiety (not shown above). The properties (activity, stability) were improved by DNA shuffling (Stemmer, 1994); in case of HHDH, the activity could be increased >2,500-fold compared to the wild-type enzyme (Fox et al., 2007).
Role of Engineered Proteins as Therapeutic Formulations
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Khushboo Gulati, Krishna Mohan Poluri
Homologous recombination techniques are based on high sequence homology of the parental gene sequences. The resultant chimeric gene shows an amalgamation of characteristic features of the combining parental sequences. The homologous recombination methods have been classified into in vitro and in vivo homologous recombination methods. DNA shuffling is the most prominent in vitro homologous recombination technique developed by Stemmer and coworkers (Stemmer, 1994b). Later advancements in the DNA shuffling method resulted in improvised schemes such as family shuffling (Crameri et al., 1998), and DOGs (degenerate oligonucleotide gene shuffling) (Gibbs et al., 2001). Other in vitro homologous recombination methods include: random priming in vitro recombination (RPR) (Shao et al., 1998), truncated metagenomic gene-specific PCR (TMGS-PCR) (Wang et al., 2010), staggered extension process (StEP) (Zhao et al., 1998), random chimeragenesis on transient templates (RACHITT) (Coco, 2003), synthetic shuffling (Ness et al., 2002). The in vivo homologous recombination methods include: cloning performed in yeast (CLERY) (Abecassis et al., 2003), Mutagenic organized recombination process by homologous in vivo grouping (MORPHING) (Gonzalez-Perez et al., 2014), and phage assisted continuous evolution (PACE) (Esvelt et al., 2011).
Affinity maturation of antibodies by combinatorial codon mutagenesis versus error-prone PCR
Published in mAbs, 2020
Jan Fredrik Simons, Yoong Wearn Lim, Kyle P. Carter, Ellen K. Wagner, Nicholas Wayham, Adam S. Adler, David S. Johnson
One major challenge of antibody affinity maturation is that comprehensively mutagenized libraries can be enormously diverse. Libraries encoding multiple combinations per clone rapidly become very large. For example, while a library comprising all possible single amino acid variants in the heavy and light chain V(D)J regions of a typical antibody with 226 amino acids would contain 4,520 unique clones, a library comprising two simultaneous changes would comprise over 10 million different clones. Such large libraries can be expensive to produce, and thus investigators rely on more economical methods, such as error-prone polymerase chain reaction (epPCR), DNA shuffling11-14 or production of smaller libraries focusing exclusively on complementarity-determining region 3 (CDR3) domains.15,16 Highly diverse libraries can also be labor intensive and costly to screen, so investigators have developed an array of high-throughput methods, such as single-chain variable fragment (scFv) phage, yeast display, antigen-binding fragment (Fab) display, and full-length antibody mammalian display.17 Full-length antibody libraries are typically smaller and closer to the drug format (i.e., a monoclonal antibody), whereas scFv libraries are typically larger and cheaper to screen.18 However, an affinity-matured scFv may not always convert into an affinity-matured monoclonal antibody.19 Despite the absence of universally adopted protocols, we are not aware of any published studies that comprehensively examine the performance of various methods for affinity maturation side-by-side.
Emerging gene therapies for cystic fibrosis
Published in Expert Review of Respiratory Medicine, 2019
Kamran M. Miah, Stephen C. Hyde, Deborah R. Gill
Intriguingly, the diffusion of rAAV vectors (serotypes 1, 2, and 5) through CF mucus in vitro was found to be significantly hampered by its viscosity, and thus mucus is a critical extracellular barrier to effective gene transfer [89]. Only a small fraction of rAAV particles could penetrate CF mucus to reach airway epithelia, and thus it is perhaps not surprising that modulation of CF mucus viscosity with a mucolytic drug (N-acetylcysteine) improved rAAV1 vector diffusion. Further to this, alternative AAV vector serotypes, particularly serotype 6 [90], was identified as capable of rapidly diffusing through CF mucus compared with AAV1 capsid. Recombinant AAV vectors based on serotype 2 were principally used in previous clinical trials [54–56], and its reduced ability to transverse CF mucus at the airway liquid surface interface may, in part, explain the disappointing outcomes. An opportunity to explore the wider range of currently available and new AAV vector serotypes is clearly warranted. In particular, AAV capsid DNA shuffling or mutagenesis followed by directed evolution strategies (see Figure 2 for an overview of approaches to AAV directed evolution) together with next-generation sequencing permit rapid development of more effective, target-specific AAV serotypes for gene therapy applications in vivo [91–93]. Several novel AAV variants show promise after directed evolution selecting for transduction of airway epithelia. Mutagenesis of the AAV cap gene (serotypes 1, 2, 4, 5, 6, 8, and 9) and DNA shuffling yielded novel AAV2H22 from directed evolution using a combined strategy – first by selecting for capsids capable of transducing porcine airway epithelia in vitro and then by selection in large airways of 6-week old pig airway epithelia in vivo [94]. The resulting rAAV vector outperformed both rAAV1 and 6 (both effective airway serotypes), and impressively, was found to rescue tracheal ASL pH, Cl− transport, and the ability to mediate bacterial killing in the CF-pig model [94]. As a note of caution, however, directed evolution of AAV variants in animal models can show optimal tropism for the species in which it was derived, and subsequent translation to humans may be poor. Therefore, the use of human-derived tissues and/or organoids is recommended to facilitate the directed evolution of novel AAV variants more relevant for more translatable gene therapy in patients [95].
Dermatophagoides spp. hypoallergens design: what has been achieved so far?
Published in Expert Opinion on Therapeutic Patents, 2020
Eduardo Santos da Silva, Carina Silva Pinheiro, Luis Gustavo Carvalho Pacheco, Neuza Maria Alcantara-Neves
Evaluation of current and future needs in a given technological area can be performed through the analysis of scientific publications; but, when potential market value of a product or service is at stake, additional analyses on companies or research institutions’ patent portfolio might add useful information regarding common features, specifications, and trends of technologies [28,29]. In that sense, we used a double-approach analysis and our first finding revealed that both, patent applications and articles for AIT, displayed an increase in deposits and publications, respectively (Figure 1(a)). However, the cumulative curve for articles showed a sharper escalation, mainly from 2014 on, in comparison to patent applications’ filing. In the case of the cumulative numbers, while 22 Dermatophagoides spp. hypoallergen related-articles were published in 13 years, patent applications by priority date reached in 17 years a volume of 12 applications. These differences may be explained possibly by the fact that some applicants did seek protection only in their respective national offices, and, therefore, the patent applications are not currently published in EPO, but articles are available in PubMed. In addition, high costs to maintain international patents may rise as an issue, mainly for some applicants from developing countries. Regardless of these differences, the aspect of the curves illustrates that Dermatophagoides spp. hypoallergenic derivatives still are an emerging technology in AIT, maybe because in the last years new ways to design them have been developed, such as (i) construction of recombinant modified allergen by site-directed mutagenesis, producing a recombinant hypoallergen whose sequence had mutations in IgE epitopes [30–33]; (ii) fusion of T-cell epitopes in the sequence of a single-recombinant protein, without B-cell epitopes [34–37]; (iii) construction of recombinant hybrid proteins, with sequences of different allergens and sometimes modification or depletion of IgE epitopes [38]; (iv) construction of recombinant proteins as cysteine variants by mutations of these residues and consequently disrupting the tridimensional structure of the allergen [39,40]; (v) pro-enzymes recombinant expression, leading to the production of enzymatic inactive allergens [41,42]; (vi) recombinant proteins with overlapping of random peptides by using in silico tools [40,43]; (vii) B-cell and T-cell epitope vaccine construction by mixture of different sizes of synthetized peptides in a formulation [44,45]; (viii) fusion of a recombinant protein with other proteins that can lead to lower IgE responses [24,39,44,46,47]; (ix) design by DNA shuffling [48,49]; and (x) more recently, tridimensional structure-based design, producing recombinant proteins with weak binding to epitopes [43,50]. The use of these different strategies for design, whether alone or jointly, constitute, therefore, the design of hypoallergens as an innovative area with potential for growth and market value [39,40,44]. Figure 1(b) summarizes a timeline of the use of these molecular biology and protein engineering techniques.