scholarly journals Self-Inactivating Alpharetroviral Vectors with a Split-Packaging Design

2010 ◽  
Vol 84 (13) ◽  
pp. 6626-6635 ◽  
Author(s):  
Julia D. Suerth ◽  
Tobias Maetzig ◽  
Melanie Galla ◽  
Christopher Baum ◽  
Axel Schambach
Keyword(s):  
Mammalian Cells ◽  
High Titer ◽  
Viral Proteins ◽  
Retroviral Vectors ◽  
Long Terminal Repeats ◽  
Rous Sarcoma ◽  
Vector Systems ◽  
Cellular Genes ◽  
Integration Sites ◽  
Transcriptional Elements

ABSTRACT Accidental insertional activation of proto-oncogenes and potential vector mobilization pose serious challenges for human gene therapy using retroviral vectors. Comparative analyses of integration sites of different retroviral vectors have elucidated distinct target site preferences, highlighting vectors based on the alpharetrovirus Rous sarcoma virus (RSV) as those with the most neutral integration spectrum. To date, alpharetroviral vector systems are based mainly on single constructs containing viral coding sequences and intact long terminal repeats (LTR). Even though they are considered to be replication incompetent in mammalian cells, the transfer of intact viral genomes is unacceptable for clinical applications, due to the risk of vector mobilization and the potentially immunogenic expression of viral proteins, which we minimized by setting up a split-packaging system expressing the necessary viral proteins in trans. Moreover, intact LTRs containing transcriptional elements are capable of activating cellular genes. By removing most of these transcriptional elements, we were able to generate a self-inactivating (SIN) alpharetroviral vector, whose LTR transcriptional activity is strongly reduced and whose transgene expression can be driven by an internal promoter of choice. Codon optimization of the alpharetroviral Gag/Pol expression construct and further optimization steps allowed the production of high-titer self-inactivating vector particles in human cells. We demonstrate proof of principle for the versatility of alpharetroviral SIN vectors for the genetic modification of murine and human hematopoietic cells at a low multiplicity of infection.

scholarly journals Toward Tightly Tuned Gene Expression Following Lentiviral Vector Transduction

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1427
Author(s):  
Audrey Page ◽  
Floriane Fusil ◽  
François-Loïc Cosset
Keyword(s):  
Gene Expression ◽  
Transgene Expression ◽  
Lentiviral Vector ◽  
Basic Research ◽  
Lentiviral Vectors ◽  
Retroviral Vectors ◽  
Clinical Applications ◽  
Long Terminal Repeats ◽  
Vector Systems ◽  
Gene Excision

Lentiviral vectors are versatile tools for gene delivery purposes. While in the earlier versions of retroviral vectors, transgene expression was controlled by the long terminal repeats (LTRs), the latter generations of vectors, including those derived from lentiviruses, incorporate internal constitutive or regulated promoters in order to regulate transgene expression. This allows to temporally and/or quantitatively control transgene expression, which is required for many applications such as for clinical applications, when transgene expression is required in specific tissues and at a specific timing. Here we review the main systems that have been developed for transgene regulated expression following lentiviral gene transfer. First, the induction of gene expression can be triggered either by external or by internal cues. Indeed, these regulated vector systems may harbor promoters inducible by exogenous stimuli, such as small molecules (e.g., antibiotics) or temperature variations, offering the possibility to tune rapidly transgene expression in case of adverse events. Second, expression can be indirectly adjusted by playing on inserted sequence copies, for instance by gene excision. Finally, synthetic networks can be developed to sense specific endogenous signals and trigger defined responses after information processing. Regulatable lentiviral vectors (LV)-mediated transgene expression systems have been widely used in basic research to uncover gene functions or to temporally reprogram cells. Clinical applications are also under development to induce therapeutic molecule secretion or to implement safety switches. Such regulatable approaches are currently focusing much attention and will benefit from the development of other technologies in order to launch autonomously controlled systems.

scholarly journals The Core Element of a CpG Island Protects Avian Sarcoma and Leukosis Virus-Derived Vectors from Transcriptional Silencing

2008 ◽  
Vol 82 (16) ◽  
pp. 7818-7827 ◽  
Author(s):  
Filip Šenigl ◽  
Jiří Plachý ◽  
Jiří Hejnar
Keyword(s):  
Binding Sites ◽  
Mammalian Cells ◽  
Cpg Island ◽  
Cpg Islands ◽  
Transcriptional Silencing ◽  
Retroviral Vectors ◽  
Core Element ◽  
Promoter Sequences ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT Unmethylated CpG islands are known to keep adjacent promoters transcriptionally active. In the CpG island adjacent to the adenosine phosphoribosyltransferase gene, the protection against transcriptional silencing can be attributed to the short CpG-rich core element containing Sp1 binding sites. We report here the insertion of this CpG island core element, IE, into the long terminal repeat of a retroviral vector derived from Rous sarcoma virus, which normally suffers from progressive transcriptional silencing in mammalian cells. IE insertion into a specific position between enhancer and promoter sequences led to efficient protection of the integrated vector from silencing and gradual CpG methylation in rodent and human cells. Individual cell clones with IE-modified reporter vectors display high levels of reporter expression for a sustained period and without substantial variegation in the cell culture. The presence of Sp1 binding sites is important for the protective effect of IE, but at least some part of the entire antisilencing capacity is maintained in IE with mutated Sp1 sites. We suggest that this strategy of antisilencing protection by the CpG island core element may prove generally useful in retroviral vectors.

scholarly journals Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity

Blood ◽  
2006 ◽  
Vol 108 (8) ◽  
pp. 2545-2553 ◽  
Author(s):  
Ute Modlich ◽  
Jens Bohne ◽  
Manfred Schmidt ◽  
Christof von Kalle ◽  
Sabine Knöss ◽  
...  
Keyword(s):  
Cell Culture ◽  
Insertional Mutagenesis ◽  
Hematopoietic Cells ◽  
Retroviral Vectors ◽  
Stable Gene ◽  
Long Terminal Repeats ◽  
Cellular Genes ◽  
In Vitro Expansion ◽  
Vector Copy Number

AbstractRetroviral vectors with long terminal repeats (LTRs), which contain strong enhancer/promoter sequences at both ends of their genome, are widely used for stable gene transfer into hematopoietic cells. However, recent clinical data and mouse models point to insertional activation of cellular proto-oncogenes as a dose-limiting side effect of retroviral gene delivery that potentially induces leukemia. Self-inactivating (SIN) retroviral vectors do not contain the terminal repetition of the enhancer/promoter, theoretically attenuating the interaction with neighboring cellular genes. With a new assay based on in vitro expansion of primary murine hematopoietic cells and selection in limiting dilution, we showed that SIN vectors using a strong internal retroviral enhancer/promoter may also transform cells by insertional mutagenesis. Most transformed clones, including those obtained after dose escalation of SIN vectors, showed insertions upstream of the third exon of Evi1 and in reverse orientation to its transcriptional orientation. Normalizing for the vector copy number, we found the transforming capacity of SIN vectors to be significantly reduced when compared with corresponding LTR vectors. Additional modifications of SIN vectors may further increase safety. Improved cell-culture assays will likely play an important role in the evaluation of insertional mutagenesis.

Conjugation of an antibody Fv fragment to a virus coat protein: cell-specific targeting of recombinant polyoma-virus-like particles

2001 ◽  
Vol 356 (3) ◽  
pp. 867-873 ◽  
Author(s):  
Kay STUBENRAUCH ◽  
Stefan GLEITER ◽  
Ulrich BRINKMANN ◽  
Rainer RUDOLPH ◽  
Hauke LILIE
Keyword(s):  
Coat Protein ◽  
Mammalian Cells ◽  
Recombinant Antibody ◽  
Specific Antigen ◽  
Antibody Fragment ◽  
Fusion Peptides ◽  
Virus Like Particles ◽  
Vector Systems ◽  
Virus Coat

The development of cell-type-specific delivery systems is highly desirable for gene-therapeutic applications. Current virus-based vector systems show broad cell specificity, which results in the need to restrict the natural tropism of these viral systems. Here we demonstrate that tumour-cell-specific virus-like particles can be functionally assembled in vitro from recombinant viral coat protein expressed in Escherichia coli. The insertion of a negatively charged peptide in the HI loop of polyoma VP1 interferes with the binding of VP1 to the natural recognition site on mammalian cells and also serves as an adapter for the coupling of antibody fragments that contain complementary charged fusion peptides. A recombinant antibody fragment of the tumour-specific anti-(Lewis Y) antibody B3 could be coupled to the mutant VP1 by engineered polyionic peptides and an additional disulphide bond. With this system an entirely recombinant cell-specific delivery system assembled in vitro could be generated that transfers genes preferentially to cells presenting the tumour-specific antigen on the cell surface.

scholarly journals Molecular cloning of cellular genes encoding retinoblastoma-associated proteins: identification of a gene with properties of the transcription factor E2F.

1992 ◽  
Vol 12 (12) ◽  
pp. 5620-5631 ◽  
Author(s):  
B Shan ◽  
X Zhu ◽  
P L Chen ◽  
T Durfee ◽  
Y Yang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
T Antigen ◽  
Transactivation Domain ◽  
Recognition Sequence ◽  
Cellular Genes ◽  
Genes Encoding ◽  
Associated Proteins ◽  
Transcription Factor E2f

The retinoblastoma protein interacts with a number of cellular proteins to form complexes which are probably crucial for its normal physiological function. To identify these proteins, we isolated nine distinct clones by direct screening of cDNA expression libraries using purified RB protein as a probe. One of these clones, Ap12, is expressed predominantly at the G1-S boundary and in the S phase of the cell cycle. The nucleotide sequence of Ap12 has features characteristic of transcription factors. The C-terminal region binds to unphosphorylated RB in regions similar to those to which T antigen binds and contains a transactivation domain. A region containing a potential leucine zipper flanked by basic residues is able to bind an E2F recognition sequence specifically. Expression of Ap12 in mammalian cells significantly enhances E2F-dependent transcriptional activity. These results suggest that Ap12 encodes a protein with properties known to be characteristic of transcription factor E2F.

Efficient Gene Transfer to Neural Stem Cells by High Titer Retroviral Vectors

2002 ◽  
pp. 297-300
Author(s):  
Hiroko Baba ◽  
Hideki Hida ◽  
Yuji Kodama ◽  
Cha-Gyun Jung ◽  
Chun-Zhen Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Neural Stem Cells ◽  
High Titer ◽  
Retroviral Vectors ◽  
Efficient Gene

scholarly journals Identification of Homeodomain Proteins, PBX1 and PREP1, Involved in the Transcription of Murine Leukemia Virus

2003 ◽  
Vol 23 (3) ◽  
pp. 831-841 ◽  
Author(s):  
Sheng-Hao Chao ◽  
John R. Walker ◽  
Sumit K. Chanda ◽  
Nathanael S. Gray ◽  
Jeremy S. Caldwell
Keyword(s):  
Murine Leukemia Virus ◽  
Kinase Inhibitors ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Viral Transcription ◽  
Long Terminal Repeats ◽  
Homeodomain Proteins ◽  
Cellular Genes ◽  
Simplex Virus ◽  
Murine Leukemia

ABSTRACT Cyclin-dependent kinase inhibitors (CDKIs) have been shown to block human immunodeficiency virus and herpes simplex virus. It is hypothesized that CDKIs block viral replication by inhibiting transcription of specific cellular genes. Here we find that three CDKIs, flavopiridol, purvalanol A, and methoxy-roscovitine, block Moloney murine leukemia virus (MLV) transcription events. Using gene expression microarray technology to examine the inhibitory effects of CDKIs, we observed a cellular gene, the pre-B-cell leukemia transcription factor 1 (Pbx1) gene, down-regulated by CDKI treatment. The PBX consensus element (PCE), TGATTGAC, is conserved in the long terminal repeats of several murine retroviruses, including Moloney MLV. Mutations in the PCE completely inhibited viral transcription whereas overexpression of PBX1 and a PBX1-associated protein, PREP1, enhanced viral transcription. The interaction between the PCE and PBX1-PREP1 proteins was confirmed by gel shift experiments. Blocking PBX1 protein synthesis resulted in a significant decrease in viral transcription. Collectively, our results represent the first work demonstrating that the homeodomain proteins PBX1 and PREP1 are cellular factors involved in Moloney MLV transcription regulation.

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