Improved alpharetrovirus-based Gag.MS2 particles for efficient and transient delivery of CRISPR-Cas9 into target cells.

DNA-modifying technologies, such as the CRISPR-Cas9 system, are promising tools in the field of gene and cell therapies. However, high and prolonged expression of DNA-modifying enzymes may cause cytotoxic and genotoxic side effects and is therefore unwanted in therapeutic approaches. Consequently, development of new and potent short-term delivery methods is of utmost importance.

Alpharetroviral self-inactivating vectors produced by a superinfection-resistant stable packaging cell line allow genetic modification of primary human T lymphocytes.

Primary human T lymphocytes represent an important cell population for adoptive immunotherapies, including chimeric-antigen and T-cell receptor applications, as they have the capability to eliminate non-self, virus-infected and tumor cells. Given the increasing numbers of clinical immunotherapy applications, the development of an optimal vector platform for genetic T lymphocyte engineering, which allows cost-effective high-quality vector productions, remains a critical goal. Alpharetroviral self-inactivating vectors (ARV) have several advantages compared to other vector platforms, including a more random genomic integration pattern and reduced likelihood for inducing aberrant splicing of integrated proviruses.

Generation of Genetically Engineered Precursor T-Cells From Human Umbilical Cord Blood Using an Optimized Alpharetroviral Vector Platform.

Retroviral engineering of hematopoietic stem cell-derived precursor T-cells (preTs) opens the possibility of targeted T-cell transfer across human leukocyte antigen (HLA)-barriers. Alpharetroviral vectors exhibit a more neutral integration pattern thereby reducing the risk of insertional mutagenesis. Cord blood-derived CD34+ cells were transduced and differentiated into preTs in vitro.

Efficient generation of gene-modified human natural killer cells via alpharetroviral vectors.

Unlabelled: Natural killer (NK) cells play an important role in tumor immunotherapy with their unique capability of killing transformed cells without the need for prior sensitization and without major histocompatibility complex (MHC)/peptide restriction. However, tumor cells can escape NK cell cytotoxicity by various tumor immune escape mechanisms. To overcome these escape mechanisms, NK cells can be modified to express chimeric antigen receptors (CARs), enhancing their tumor-specific cytotoxicity.

RETRACTED: Novel and safer self-inactivating vectors for gene therapy of Wiskott-Aldrich Syndrome.

The article entitled “Novel and safer self-inactivating vectors for gene therapy of Wiskott-Aldrich Syndrome”, by E. G. Coci1, T.

Advantages and applications of CAR-expressing natural killer cells.

In contrast to donor T cells, natural killer (NK) cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD). In order to improve cytotoxicity against resistant cancer cells, auspicious efforts have been made with chimeric antigen receptor (CAR) expressing T- and NK cells. These CAR-modified cells express antigen receptors against tumor-associated surface antigens, thus redirecting the effector cells and enhancing tumor-specific immunosurveillance.

Alpharetroviral vectors: from a cancer-causing agent to a useful tool for human gene therapy.

Gene therapy using integrating retroviral vectors has proven its effectiveness in several clinical trials for the treatment of inherited diseases and cancer. However, vector-mediated adverse events related to insertional mutagenesis were also observed, emphasizing the need for safer therapeutic vectors. Paradoxically, alpharetroviruses, originally discovered as cancer-causing agents, have a more random and potentially safer integration pattern compared to gammaretro- and lentiviruses.

Genome-wide analysis of alpharetroviral integration in human hematopoietic stem/progenitor cells.

Gene transfer vectors derived from gamma-retroviruses or lentiviruses are currently used for the gene therapy of genetic or acquired diseases. Retroviral vectors display a non-random integration pattern in the human genome, targeting either regulatory regions (gamma-retroviruses) or the transcribed portion of expressed genes (lentiviruses), and have the potential to deregulate gene expression at the transcriptional or post-transcriptional level. A recently developed alternative vector system derives from the avian sarcoma-leukosis alpha-retrovirus (ASLV) and shows favorable safety features compared to both gamma-retroviral and lentiviral vectors in preclinical models.

Modified lentiviral LTRs allow Flp recombinase-mediated cassette exchange and in vivo tracing of "factor-free" induced pluripotent stem cells.

Methods for generating induced pluripotent stem cells (iPSCs) for disease modeling and cell therapies have progressed from integrating vectors to transient delivery of reprogramming factors, avoiding permanent genomic modification. A major limitation of unmodified iPSCs is the assessment of their distribution and contribution to adverse reactions in autologous cell therapy. Here, we report that polycistronic lentiviral vectors with single Flp recombinase (Flp) recognition target (FRT) sites can be used to generate murine iPSCs that are devoid of the reprogramming cassette but carry an intergenic 300-bp long terminal repeat sequence.

Deletion of the LTR enhancer/promoter has no impact on the integration profile of MLV vectors in human hematopoietic progenitors.

Moloney murine leukemia virus (MLV)-derived gamma-retroviral vectors integrate preferentially near transcriptional regulatory regions in the human genome, and are associated with a significant risk of insertional gene deregulation. Self-inactivating (SIN) vectors carry a deletion of the U3 enhancer and promoter in the long terminal repeat (LTR), and show reduced genotoxicity in pre-clinical assays. We report a high-definition analysis of the integration preferences of a SIN MLV vector compared to a wild-type-LTR MLV vector in the genome of CD34(+) human hematopoietic stem/progenitor cells (HSPCs).

Alpharetroviral vector-mediated gene therapy for X-CGD: functional correction and lack of aberrant splicing.

Comparative integrome analysis has revealed that the most neutral integration pattern among retroviruses is attributed to alpharetroviruses. We chose X-linked chronic granulomatous disease (X-CGD) as model to evaluate the potential of self-inactivating (SIN) alpharetroviral vectors for gene therapy of monogenic diseases. Therefore, we combined the alpharetroviral vector backbone with the elongation factor-1α short promoter, both considered to possess a low genotoxic profile, to drive transgene (gp91(phox)) expression.

Genetic modification of lymphocytes by retrovirus-based vectors.

The genetic modification of lymphocytes is an important topic in the emerging field of gene therapy. Many clinical trials targeting immunodeficiency syndromes or cancer have shown therapeutic benefit; further applications address inflammatory and infectious disorders. Retroviral vector development requires a detailed understanding of the interactions with the host.

Alpharetroviral self-inactivating vectors: long-term transgene expression in murine hematopoietic cells and low genotoxicity.

Comparative integrome analyses have highlighted alpharetroviral vectors with a relatively neutral, and thus favorable, integration spectrum. However, previous studies used alpharetroviral vectors harboring viral coding sequences and intact long-terminal repeats (LTRs). We recently developed self-inactivating (SIN) alpharetroviral vectors with an advanced split-packaging design.