This disclosure provides modified recombinant retroviruses comprisings a transgene encoding a protein with a heterologous secretion signal, containing a 2A-peptide or peptide-like coding sequence operably linked to a heterologous polynucleotide, The disclosure further relates to cells and vector expressing or comprising such vectors and methods of using such modified vectors in the treatment of disease and disorders.

This disclosure provides modified recombinant retroviruses comprisings containing a 2A-peptide or peptide-like coding sequence operably linked to a heterologous polynucleotide. The disclosure further relates to cells and vector expressing or comprising such vectors and methods of using such modified vectors in the treatment of disease and disorders.

Type-C endogenous retroviruses (ERVs) embedded in Chinese hamster ovary (CHO) cells were altered to modify the release of retroviral and/or retroviral-like particles in the culture supernatant. Although evidence for the infectivity of these particles is missing, their presence has raised safety concerns. 173 type-C ERV sequences that clustered into functionally conserved groups were identified. Transcripts from one type-C ERV group were identified to be full-length with intact open reading frames, and to have corresponding viral RNA genomes that were loaded into retroviral-like particles. Also, sequence analysis of the genomic RNA from viral particles indicated that they may result from few expressed ERV sequences. Disclosed herein is the disruption/alteration of the gag gene of the expressed ERV group using CRISPR-Cas9 genome editing. Comparison of CRISPR-derived mutations at the DNA and mRNA level led to the identification of a single ERV locus responsible for the release of viral RNA-loaded particles from CHO cells. Clones bearing a Gag loss-of-function mutation in this particular ERV locus showed a reduction of viral RNA-containing particles in the cell supernatant by over 250-fold. Notably, ERV mutagenesis did not compromise cell growth, cell size or recombinant protein production. Provided herein is a new strategy and cells, in particular engineered CHO cells, to mitigate potential contaminations from CHO endogenous retroviruses during biopharmaceutical manufacturing.

A strong insulator fragment from foamy virus, which can be used to insulate expression of a transgene and reduce genotoxicity of integrating vectors comprising such. The insulator fragment can also be used in gene targeting constructs in gene editing.

Described herein are uses of fluorescent enveloped virus particles as standards in nanoscale flow cytometry applications. The virus particles comprise a fluorescent dye or a fluorescent protein. A standard ladder comprising a plurality of fluorescent enveloped virus particles of different sizes is also provided. The standards may also comprise marker(s) of interest, and may be used as controls for detection of other viruses or extracellular vesicle, e.g. in diagnostic applications. Methods of producing controls for such applications are provided, including those having desired profiles. The controls may be used for enumeration of markers on microparticles (e.g. extracellular vesicles or viruses). Also described is a modified gammaretrovirus comprising a mutation that abolishes expression of the viral glyco-Gag protein, and having a fluorescent protein inserted in-frame into the proline-rich region (PRR) of the viral env protein. The gammaretrovirus may be M-MLV bearing a mutation that abolishes expression of the glyco-Gal protein, gPr80.

This disclosure provides modified recombinant retroviruses comprisings containing a 2A-peptide or peptide-like coding sequence operably linked to a heterologous polynucleotide. The disclosure further relates to cells and vector expressing or comprising such vectors and methods of using such modified vectors in the treatment of disease and disorders.

Disclosed herein are compositions and methods of making and using bat IPSCs (BipS). Also disclosed herein are methods and compositions of virus nucleic acids residing in bat IPSCs. Also disclosed are nucleotides, cells, and methods associated with the compositions including their use as vaccines.

Type-C endogenous retroviruses (ERVs) embedded in Chinese hamster ovary (CHO) cells were altered to modify the release of retroviral and/or retroviral-like particles in the culture supernatant. Although evidence for the infectivity of these particles is missing, their presence has raised safety concerns. 173 type-C ERV sequences that clustered into functionally conserved groups were identified. Transcripts from one type-C ERV group were identified to be full-length with intact open reading frames, and to have corresponding viral RNA genomes that were loaded into retroviral-like particles. Also, sequence analysis of the genomic RNA from viral particles indicated that they may result from few expressed ERV sequences. Disclosed herein is the disruption/alteration of the gag gene of the expressed ERV group using CRISPR-Cas9 genome editing. Comparison of CRISPR-derived mutations at the DNA and mRNA level led to the identification of a single ERV locus responsible for the release of viral RNA-loaded particles from CHO cells. Clones bearing a Gag loss-of-function mutation in this particular ERV locus showed a reduction of viral RNA-containing particles in the cell supernatant by over 250-fold. Notably, ERV mutagenesis did not compromise cell growth, cell size or recombinant protein production. Provided herein is a new strategy and cells, in particular engineered CHO cells, to mitigate potential contaminations from CHO endogenous retroviruses during biopharmaceutical manufacturing.

Type-C endogenous retroviruses (ERVs) embedded in Chinese hamster ovary (CHO) cells were altered to modify the release of retroviral and/or retroviral-like particles in the culture supernatant. Although evidence for the infectivity of these particles is missing. their presence has raised safety concerns. 173 type-C ERV sequences that clustered into functionally conserved groups were identified. Transcripts from one type-C ERV group were identified to be full-length with intact open reading frames, and to have corresponding viral RNA genomes that were loaded into retroviral-like particles. Also, sequence analysis of the genomic RNA from viral particles indicated that they may result from few expressed ERV sequences. Disclosed herein is the disruption/alteration of the gag gene of the expressed ERV group using CRISPR-Cas9 genome editing. Comparison of CRISPR-derived mutations at the DNA and mRNA level led to the identification of a single ERV locus responsible for the release of viral RNA-loaded particles from CHO cells. Clones bearing a Gag loss-of-function mutation in this particular ERV locus showed a reduction of viral RNA-containing particles in the cell supernatant by over 250-fold. Notably, ERV mutagenesis did not compromise cell growth, cell size or recombinant protein production. Provided herein is a new strategy and cells, in particular engineered CHO cells, to mitigate potential contaminations from CHO endogenous retroviruses during biopharmaceutical manufacturing.