Peptide Ligands Incorporated into the Threefold Spike Capsid Domain to Re-Direct Gene Transduction of AAV8 and AAV9 In Vivo

Efficiency and specificity of viral vectors are vital issues in gene therapy. Insertion of peptide ligands into the adeno-associated viral (AAV) capsid at receptor binding sites can re-target AAV2-derived vectors to alternative cell types. Also, the use of serotypes AAV8 and -9 is more efficient than AAV2 for gene transfer to certain tissues in vivo. Consequently, re-targeting of these serotypes by ligand insertion could be a promising approach but has not been explored so far. Here, we generated AAV8 and -9 vectors displaying peptides in the threefold spike capsid domain. These peptides had been selected from peptide libraries displayed on capsids of AAV serotype 2 to optimize systemic gene delivery to murine lung tissue and to breast cancer tissue in PymT transgenic mice (PymT). Such peptide insertions at position 590 of the AAV8 capsid and position 589 of the AAV9 capsid changed the transduction properties of both serotypes. However, both peptides inserted in AAV8 did not result in the same changes of tissue tropism as they did in AAV2. While the AAV2 peptides selected on murine lung tissue did not alter tropism of serotypes 8 and -9, insertion of the AAV2-derived peptide selected on breast cancer tissue augmented tumor gene delivery in both serotypes. Further, this peptide mediated a strong but unspecific in vivo gene transfer for AAV8 and abrogated transduction of various control tissues for AAV9. Our findings indicate that peptide insertion into defined sites of AAV8 and -9 capsids can change and improve their efficiency and specificity compared to their wild type variants and to AAV2, making these insertion sites attractive for the generation of novel targeted vectors in these serotypes

Recombinant Production of MFHR1, A Novel Synthetic Multitarget Complement Inhibitor, in Moss Bioreactors

The human complement system is an important part of the immune system responsible for lysis and elimination of invading microorganisms and apoptotic body cells. Improper activation of the system due to deficiency, mutations, or autoantibodies of complement regulators, mainly factor H (FH) and FH-related proteins (FHRs), causes severe kidney and eye diseases. However, there is no recombinant FH therapeutic available on the market. The first successful recombinant production of FH was accomplished with the moss bioreactor, Physcomitrella patens. Recently, a synthetic regulator, MFHR1, was designed to generate a multitarget complement inhibitor that combines the activities of FH and the FH-related protein 1 (FHR1). The potential of MFHR1 was demonstrated in a proof-of-concept study with transiently transfected insect cells. Here, we present the stable production of recombinant glyco-engineered MFHR1 in the moss bioreactor. The key features of this system are precise genome engineering via homologous recombination, Good Manufacturing Practice-compliant production in photobioreactors, high batch-to-batch reproducibility, and product stability. Several potential biopharmaceuticals are being produced in this system. In some cases, these are even biobetters, i.e., the recombinant proteins produced in moss have a superior quality compared to their counterparts from mammalian systems as for example moss-made aGal, which successfully passed phase I clinical trials. Via mass spectrometry-based analysis of moss-produced MFHR1, we now prove the correct synthesis and modification of this glycoprotein with predominantly complex-type N-glycan attachment. Moss-produced MFHR1 exhibits cofactor and decay acceleration activities comparable to FH, and its mechanism of action on multiple levels within the alternative pathway of complement activation led to a strong inhibitory activity on the whole alternative pathway, which was higher than with the physiological regulator FH

Successful Expansion but Not Complete Restriction of Tropism of Adeno-Associated Virus by In Vivo Biopanning of Random Virus Display Peptide Libraries

Targeting viral vectors to certain tissues in vivo has been a major challenge in gene therapy. Cell type-directed vector capsids can be selected from random peptide libraries displayed on viral capsids in vitro but so far this system could not easily be translated to in vivo applications. Using a novel, PCR-based amplification protocol for peptide libraries displayed on adeno-associated virus (AAV), we selected vectors for optimized transduction of primary tumor cells in vitro. However, these vectors were not suitable for transduction of the same target cells under in vivo conditions. We therefore performed selections of AAV peptide libraries in vivo in living animals after intravenous administration using tumor and lung tissue as prototype targets. Analysis of peptide sequences of AAV clones after several rounds of selection yielded distinct sequence motifs for both tissues. The selected clones indeed conferred gene expression in the target tissue while gene expression was undetectable in animals injected with control vectors. However, all of the vectors selected for tumor transduction also transduced heart tissue and the vectors selected for lung transduction also transduced a number of other tissues, particularly and invariably the heart. This suggests that modification of the heparin binding motif by target-binding peptide insertion is necessary but not sufficient to achieve tissue-specific transgene expression. While the approach presented here does not yield vectors whose expression is confined to one target tissue, it is a useful tool for in vivo tissue transduction when expression in tissues other than the primary target is uncritical

Selektion und Charakterisierung zielgerichteter Vektorkapside aus randomisierten AAV-2 Peptidbanken

Selection of viral vectors by screening viral display peptide libraries is an auspicious approach to improve safety and efficiency of gene vectors. The screening of random AAV peptide libraries occurs via the amplification of viruses from a multitude of potential targeting peptides each presented within an AAV capsid that are internalized into target cells, mediated by the peptide displayed on their surface.The aim of this thesis was the selection of cell type- or tissue-directed gene vectors from random peptide libraries displayed on adeno-associated virus (AAV) and their characterization.Immature malignant blood progenitor cells causing acute myeloid leukemia (AML) are generally considered to be transduction-resistant to most conventional gene vectors. We screened random AAV serotype 2 peptide libraries on AML cells to select vector capsids with optimized leukemia transduction capacity. The screening revealed a distinct peptide sequence motif displayed on the selected viral capsids. The capsid mutant displaying the peptide NQVGSWS transduced the leukemia cell line Kasumi-1 with up to 90% efficiency, in contrast to vectors displaying a random unselected peptide (0.2% efficiency). Transduction assays on a panel of cell lines showed that the NQVGSWS capsid was able to overcome resistance to AAV-transduction especially in hematopoietic cancer cells. We further showed that NQVGSWS transduction of leukemia cells is independent of the primary attachment receptor heparin sulfate proteoglycan that is used for infection by wild-type AAV-2. Finally, leukemia targeted NQVGSWS-AAV vectors harboring a suicide gene conferred selective killing to Kasumi-1 AML cells. Therefore, we concluded that the selected vector capsids are a suitable and valuable tool to target therapeutic genes to AML cells.Screening AAV peptide libraries in vivo provides much more appropriate conditions to select for tissue-targeted gene vectors than mere cell-based in vitro approaches. In the second part of this thesis we developed a PCR-based amplification method allowing for adenovirus independent screening of AAV libraries. We performed in vivo selections applying several kinetic approaches in animals over multiple rounds after intravenous administration. The polyoma middle T-transgenic murine breast cancer and murine lung tissue were used as prototype targets. The peptide sequences of AAV clones yielded distinct sequence motifs unique for the target tissue. Selected capsid mutants conferred gene expression in the target tissue which was not detectable in animals injected with control vectors. However, most of the clones also transduced heart tissue in addition to the target tissue. We therefore conclude that this approach may be particularly useful if the tropism of the intended gene transfer in vivo has to be extended to rather than confined to the tissue of interest, indicating that targeting AAV to certain tissues in vivo seem to require more than one capsid modification. This impact the further development and improvement of AAV peptide libraries.Taken together, the work presented here demonstrates that random AAV displayed peptide libraries can be used to select for improved gene delivery vectors in vitro and, which is entirely novel, in vivo. Our results broaden the knowledge of transduction behavior of vectors isolated from AAV-2 libraries on different targets in vitro and in vivo and showed that such vectors have the potential to be used for therapeutic gene transfer

Factors Affecting Prayer

https://digitalcommons.andrews.edu/sem-videos/1015/thumbnail.jp

Novel Cytotoxic Vectors Based on Adeno-Associated Virus

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