Characterization of a new IN-I-PpoI fusion protein and a homology-arm containing transgene cassette that improve transgene expression persistence and 28S rRNA gene-targeted insertion of lentiviral vectors.

Targeting transgene integration into a safe genomic locus would be very important for gene therapy. We have generated lentivirus vectors containing the ribosomal RNA-recognising I-PpoI endonuclease fused to viral integrase, and transgene cassettes with target site homology arms to enhance insertion targeting. These new vectors were characterised with respect to the persistence of transgene expression, insertion targeting efficiency and chromosomal integrity of the transduced cells. The aim was to find an optimally safe and effective vector for human gene therapy. Fusion protein vectors with high endonuclease activity were the most effective in the accurate targeting of transgene insertion. The homology construct increased the insertion targeting efficiency to 28% in MRC-5 cells. However, karyotyping analysis showed that the high endonuclease activity induced the formation of derivative chromosomes in as many as 24% of the analysed primary T lymphocytes. The persistence of transgene expression was excellent in homology arm-containing fusion protein vectors with reduced endonuclease activity, and these fusion proteins did not cause any detectable chromosomal rearrangements attributable to the endonuclease activity. We thus conclude that instead of the fusion protein vectors that carry a highly active endonuclease, our vectors with the ability to tether the lentivirus preintegration complex to benign loci in the genome without high ribosomal DNA cleavage activity are better suited for lentivirus-based gene therapy applications.

Using the Jurkat reporter T cell line for evaluating the functionality of novel chimeric antigen receptors.

Background: T cells that are genetically modified with chimeric antigen receptor (CAR) hold promise for immunotherapy of cancer. Currently, there are intense efforts to improve the safety and efficacy of CAR T cell therapies against liquid and solid tumors. Earlier we designed a novel CAR backbone (FiCAR) where the spacer is derived from immunoglobulin (Ig) -like domains of the signal-regulatory protein alpha (SIRPα). However, the analysis of novel CAR using primary T cells is slow and laborious. Methods: To explore the versatility of the CAR backbone, we designed a set of variant FiCARs with different spacer lengths and targeting antigens. To expedite the analysis of the novel CARs, we transduced the FiCAR genes using lentiviruses into Jurkat reporter T cells carrying fluorescent reporter genes. The expression of fluorescent markers in response to FiCAR engagement with targets was analyzed by flow cytometry, and cytotoxicity was evaluated using killing assays. Furthermore, the killing mechanisms that are employed by FiCAR-equipped Jurkat T cells were investigated by flow cytometry, and the intracellular pathways involved in signaling by FiCAR were analyzed by phosphoproteomic analysis using mass spectrometry. Results: Seven different CARs were designed and transduced into Jurkat reporter cells. We show that the SIRPα derived FiCARs can be detected by flow cytometry using the SE12B6A4 antibody recognizing SIRPα. Furthermore, FiCAR engagement leads to robust activation of NFκß and NFAT signaling, as demonstrated by the expression of the fluorescent reporter genes. Interestingly, the Jurkat reporter system also revealed tonic signaling by a HER-2 targeting FiCAR. FiCAR-equipped Jurkat T cells were cytotoxic in cocultures with target cells and target cell engagement lead to an upregulation of CD107a on the Jurkat reporter T cell surface. Phosphoproteomic analyses confirmed signal transduction via the intracellular CD28/CD3ζ sequences upon the interaction of the FiCAR1 with its antigen. In addition, downstream signaling of CD3ζ/ZAP70- SLP-76-PLCγ, PI3K-AKT-NFκB pathways and activation of NFAT and AP-1 were observed. Conclusion: We conclude that the FiCAR backbone can be shortened and lengthened at will by engineering it with one to three SIRPα derived Ig-like domains, and the FiCARs are functional when equipped with different single chain variable fragment target binding domains. The Jurkat reporter system expedites the analysis of novel CARs as to their expression, signaling function, evaluation of tonic signaling issues and cytotoxic activity.

Novel modular chimeric antigen receptor spacer for T cells derived from signal regulatory protein alpha Ig-like domains.

Background: T cells equipped with chimeric antigen receptors (CAR) have shown remarkable efficacy in targeting B lineage malignancies. Improvement of the CAR structure is needed, however, with a view to developing flexibly modifiable spacers that are inert in interactions with unwanted cells. Specifically, binding to cells carrying receptors for IgG's crystallizable fragment (FcR), that recognize IgG-derived domains in CARs is to be avoided. Methods: Two novel CARs targeting the CD19 antigen where the IgG1-CH2 and -CH3 domains were replaced with Ig-like domains from signal-regulatory protein α (SIRPα) were designed in silico. An IgG1-based CAR and a CAR lacking both SIRPα and IgG1 domains were used as comparators. The phenotype and memory phenotype of the expanded cells were analyzed by flow cytometry, and CAR T cell activation and cytotoxic efficacy were assessed in co-culture experiments in response to CD19+ target cells. Unwanted interactions with FcR-expressing myeloid cells were interrogated in co-culture assays with THP-1 monocytic cells. Results: T cells carrying the novel SIRPα-based CARs enacted potent in vitro cytotoxicity against CD19 positive B-lineage leukemia cells, comparable to traditional IgG1-based CAR T cells. Co-culture of IgG1-based CAR T cells with FcR-expressing THP-1 monocytic cells led to prominent cell surface expression of CD69 on T cells together with production of Interleukin (IL)-2 and Interferon-γ, and production of IL-1ß, indicating activation of the T cells and monocytes, respectively. Longer co-culture led to killing of the monocytes. No signs of T cell nor monocyte activation were detected in co-cultures of SIRPα-based CAR T cells with THP-1 cells. Arming T cells with the SIRPα-based CARs favored differentiation towards CD4+ phenotype during expansion, while the effects on memory phenotype of the T cells were equivalent between the SIRPα- and IgG1-based CARs. In a pilot experiment, T cells modified with one of the SIRPα-based CARs showed dose dependent leukemia cell control. Conclusion: The novel SIRPα based spacers offer a suitable backbone for developing chimeric antigen receptors that evade the off-target binding to FcR while the cells retain a favorable memory phenotype and efficient cytotoxicity, establishing a promising candidate for future in vivo and clinical testing.

Efficient Nuclease-Directed Integration of Lentivirus Vectors into the Human Ribosomal DNA Locus.

Lentivirus vectors (LVs) are efficient tools for gene transfer, but the non-specific nature of transgene integration by the viral integration machinery carries an inherent risk for genotoxicity. We modified the integration machinery of LVs and harnessed the cellular DNA double-strand break repair machinery to integrate transgenes into ribosomal DNA, a promising genomic safe-harbor site for transgenes. LVs carrying modified I-PpoI-derived homing endonuclease proteins were characterized in detail, and we found that at least 21% of all integration sites localized to ribosomal DNA when LV transduction was coupled to target DNA cleavage. In addition to the primary sequence recognized by the endonuclease, integration was also enriched in chromatin domains topologically associated with nucleoli, which contain the targeted ribosome RNA genes. Targeting of this highly repetitive region for integration was not associated with detectable DNA deletions or negative impacts on cell health in transduced primary human T cells. The modified LVs characterized here have an overall lower risk for insertional mutagenesis than regular LVs and can thus improve the safety of gene and cellular therapy.

Development of Lentiviral Vectors for Targeted Integration and Protein Delivery.

The method in this chapter describes the design of human immunodeficiency virus type 1 (HIV-1) integrase (IN)-fusion proteins which we have developed to transport different proteins into the nuclei of lentiviral vector (LV)-transduced cells. The IN-fusion protein cDNA is incorporated into the LV packaging plasmid, which leads to its incorporation into vector particles as part of a large Gag-Pol polyprotein. This specific feature of protein packaging enables also the incorporation of cytotoxic and proapoptotic proteins, such as frequently cutting endonucleases and P53. The vectors can hence be used for various protein transduction needs. An outline of the necessary methods is also given to study the functionality of a chosen IN-fusion protein in a cell culture assay.

Lentiviral protein transduction with genome-modifying HIV-1 integrase-I-PpoI fusion proteins: studies on specificity and cytotoxicity.

Rare-cutting endonucleases, such as the I-PpoI, can be used for the induction of double strand breaks (DSBs) in genome editing and targeted integration based on homologous recombination. For therapeutic approaches, the specificity and the pattern of off-target effects are of high importance in these techniques. For its applications, the endonuclease needs to be transported into the target cell nucleus, where the mechanism of transport may affect its function. Here, we have studied the lentiviral protein transduction of the integrase (IN)-PpoI fusion protein using the cis-packaging method. In genome-wide interaction studies, IN-fusion proteins were verified to bind their target sequence containing 28S ribosomal RNA (rRNA) genes with a 100-fold enrichment, despite the well-documented behavior of IN to be tethered into various genomic areas by host-cell factors. In addition, to estimate the applicability of the method, DSB-induced cytotoxic effects with different vector endonuclease configurations were studied in a panel of cells. Varying the amount and activity of endonuclease enabled the adjustment of ratio between the induced DSBs and transported DNA. In cell studies, certain cancerous cell lines were especially prone to DSBs in rRNA genes, which led us to test the protein transduction in a tumour environment in an in vivo study. In summary, the results highlight the potential of lentiviral vectors (LVVs) for the nuclear delivery of endonucleases.

rDNA-directed integration by an HIV-1 integrase--I-PpoI fusion protein.

Integrating viral vectors are efficient gene transfer tools, but their integration patterns have been associated with genotoxicity and oncogenicity. The recent development of highly specific designer nucleases has enabled target DNA modification and site-specific gene insertion at desired genomic loci. However, a lack of consensus exists regarding a perfect genomic safe harbour (GSH) that would allow transgenes to be stably and reliably expressed without adversely affecting endogenous gene structure and function. Ribosomal DNA (rDNA) has many advantages as a GSH, but efficient means to target integration to this locus are currently lacking. We tested whether lentivirus vector integration can be directed to rDNA by using fusion proteins consisting of the Human Immunodeficiency Virus 1 (HIV-1) integrase (IN) and the homing endonuclease I-PpoI, which has natural cleavage sites in the rDNA. A point mutation (N119A) was introduced into I-PpoI to abolish unwanted DNA cleavage by the endonuclease. The vector-incorporated IN-I-PpoIN119A fusion protein targeted integration into rDNA significantly more than unmodified lentivirus vectors, with an efficiency of 2.7%. Our findings show that IN-fusion proteins can be used to modify the integration pattern of lentivirus vectors, and to package site-specific DNA-recognizing proteins into vectors to obtain safer transgene integration.

Production of HIV-1 integrase fusion protein-carrying lentiviral vectors for gene therapy and protein transduction.

Lentiviral vectors have broad target cell tropism and efficient machinery to integrate transgenes into the host genome. Modification of these vectors by incorporating heterologous proteins into virions has relied mostly on the fusion of proteins into the HIV-1 accessory protein Vpr. Vpr expression can be harmful for cells and its gene has been deleted from third-generation vector production plasmids. We therefore developed a direct integrase fusion protein strategy as an alternative way to package heterologous proteins into vectors. The method was tested by creating two different integrase fusion proteins, IN-p53 and IN-mCherry, cloned into the 3' end of pol in the packaging plasmid. Lentiviral vectors were produced by conventional methods, using the modified packaging plasmids. Vector-incorporated fusion proteins were correctly processed from Gag-Pol, retained the ability to catalyze transgene integration, and showed fusion protein-specific activity by being fluorescent or inducing apoptosis. Functional third-generation lentiviral vectors containing IN-fusion proteins can thus be produced by standard production protocols independent of Vpr expression. Our results suggest that this packaging method is useful for lentiviral vector-mediated protein transduction, such as intranuclear meganuclease, transposon, or zinc finger protein delivery, intracellular imaging of vector particles, and generation of modified lentiviral vectors that contain both toxic and nontoxic IN-fusion proteins.

Challenges in monoclonal antibody-based therapies.

Therapeutic monoclonal antibodies (mAbs) are the fastest growing class of new therapeutic molecules. They hold great promises for the treatment of a variety of diseases, including chronic inflammatory diseases and cancer. However, the current manufacturing and purification processes cause limitations in the production capacity of therapeutic antibodies, leading to an increase in cost. Genetic delivery of therapeutic monoclonal antibodies by in vivo production offers a new potential solution to these problems. Firstly, therapeutic efficacy can be improved by maintaining stable therapeutic, non-toxic levels within the blood circulation over a long period of time. Repeated high-dose bolus injections could be avoided, thereby reducing the possibility of side-effects. Secondly, the high cost of manufacturing and purification of the therapeutic antibodies could be reduced, making an in vivo/ex vivo mAb gene transfer an economically viable and attractive option. In general, three approaches can be used for the stable long-term expression and secretion of therapeutic antibodies in vivo: 1) direct in vivo administration of integrating vectors carrying a mAb gene, 2) grafting of ex vivo genetically modified autologous cells, and 3) implantation of an encapsulated antibody producing heterologous or autologous cells. This paper describes the key factors and problems associated with the current antibody-based immunotherapies and reviews prospects for genetic in vivo delivery of therapeutic antibodies.

A multipurpose vector system for the screening of libraries in bacteria, insect and mammalian cells and expression in vivo.

We have constructed a novel tetra-promoter vector (pBVboostFG) system that enables screening of gene/cDNA libraries for functional genomic studies. The vector enables an all-in-one strategy for gene expression in mammalian, bacterial and insect cells and is also suitable for direct use in vivo. Virus preparation is based on an improved mini Tn7 transpositional system allowing easy and fast production of recombinant baculoviruses with high diversity and negligible background. Cloning of the desired DNA fragments or libraries is based on the recombination system of bacteriophage lambda. As an example of the utility of the vector, genes or cDNAs of 18 different proteins were cloned into pBVboostFG and expressed in different hosts. As a proof-of-principle of using the vector for library screening, a chromophoric Thr65-Tyr-Gly67-stretch of enhanced green fluorescent protein was destroyed and subsequently restored by novel PCR strategy and library screening. The pBVboostFG enables screening of genome-wide libraries, thus making it an efficient new platform technology for functional genomics.