Production and Application of Multicistronic Constructs for Various Human Disease Therapies

Shaimardanova, Alisa A.; Chulpanova, Daria S.; Kitaeva, Kristina V.; Abdrakhmanova, Ilmira I.; Чернов, В. М.; Rutland, Catrin S.; Rizvanov, Albert A.; Solovyeva, Valeriya V.

doi:10.3390/pharmaceutics11110580

Cited by 32 publications

(32 citation statements)

References 100 publications

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“…However, with the exception of the rSA11/NSP3-fS1, all the viruses generated more 2A-cleaved S product than read-through product. Mutation of residues in and around the 2A element, including the inclusion of flexible linker sequences, may decrease the relative frequency of read through [ 60 , 61 ].…”

Section: Resultsmentioning

confidence: 99%

Rotavirus as an Expression Platform of Domains of the SARS-CoV-2 Spike Protein

Philip

Patton

2021

Vaccines

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Among vaccines administered to children are those targeting rotavirus, a segmented double-stranded RNA virus that represents a major cause of severe gastroenteritis. To explore the feasibility of establishing a combined rotavirus-SARS-CoV-2 vaccine, we generated recombinant (r)SA11 rotaviruses with modified segment 7 RNAs that contained coding cassettes for NSP3, a translational 2A stop-restart signal, and a FLAG-tagged portion of the SARS-CoV-2 spike (S) protein: S1 fragment, N-terminal domain (NTD), receptor-binding domain (RBD), extended RBD (ExRBD), or S2 core (CR) domain. Generation of rSA11 containing the S1 coding sequence required a sequence insertion of 2.2 kbp, the largest such insertion yet introduced into the rotavirus genome. Immunoblotting showed that rSA11 viruses containing the smaller NTD, RBD, ExRBD, and CR coding sequences expressed S-protein products of expected size, with ExRBD expressed at highest levels. These rSA11 viruses were genetically stable during serial passage. In contrast, the rSA11 virus containing the full-length S coding sequence (rSA11/NSP3-fS1) failed to express its expected 80 kDa fS1 product, for unexplained reasons. Moreover, rSA11/NSP3-fS1 was genetically unstable, with variants lacking the S1 insertion appearing during serial passage. Nonetheless, these results emphasize the potential usefulness of rotavirus vaccines as expression vectors of immunogenic portions of the SARS-CoV-2 S protein, including NTD, RBD, ExRBD, and CR, that have sizes smaller than the S1 fragment.

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Section: Resultsmentioning

confidence: 99%

Rotavirus as an Expression Platform of Domains of the SARS-CoV-2 Spike Protein

Philip

Patton

2021

Vaccines

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“…Generally speaking, pDNAs used for in vitro transfection studies vary not only in the specific reporter gene sequence they were endowed with (e.g., firefly luciferase, green fluorescence protein (GFP)), but also in other basic elements. An illustrative example of this issue relies on multicistronic constructs (i.e., pDNAs that express multiple genes at once, leading to the production of two or more separate proteins from the same mRNA) [84]. Alongside this, a specific promoter must be carefully selected in order to drive the constitutive overexpression of the transgene(s) in the specific host.…”

Section: Plasmid Dnamentioning

confidence: 99%

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

et al. 2020

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Transfection by means of non-viral gene delivery vectors is the cornerstone of modern gene delivery. Despite the resources poured into the development of ever more effective transfectants, improvement is still slow and limited. Of note, the performance of any gene delivery vector in vitro is strictly dependent on several experimental conditions specific to each laboratory. The lack of standard tests has thus largely contributed to the flood of inconsistent data underpinning the reproducibility crisis. A way researchers seek to address this issue is by gauging the effectiveness of newly synthesized gene delivery vectors with respect to benchmarks of seemingly well-known behavior. However, the performance of such reference molecules is also affected by the testing conditions. This survey points to non-standardized transfection settings and limited information on variables deemed relevant in this context as the major cause of such misalignments. This review provides a catalog of conditions optimized for the gold standard and internal reference, 25 kDa polyethyleneimine, that can be profitably replicated across studies for the sake of comparison. Overall, we wish to pave the way for the implementation of standardized protocols in order to make the evaluation of the effectiveness of transfectants as unbiased as possible.

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Section: Expression Of S Coding Sequences By Rsa11 Rotavirusesmentioning

confidence: 99%

Rotavirus as an Expression Platform of the SARS-CoV-2 Spike Protein

Philip

Patton

2021

Preprint

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Rotavirus, a segmented double-stranded RNA virus, is a major cause of acute gastroenteritis in young children. The introduction of live oral rotavirus vaccines has reduced the incidence of rotavirus disease in many countries. To explore the possibility of establishing a combined rotavirus-SARS-CoV-2 vaccine, we generated recombinant (r)SA11 rotaviruses with modified segment 7 RNAs that contained coding sequences for NSP3 and FLAG-tagged portions of the SARS-CoV-2 spike (S) protein. A 2A translational element was used to drive separate expression of NSP3 and the S product. rSA11 viruses were recovered that encoded the S-protein S1 fragment, N-terminal domain (NTD), receptor-binding domain (RBD), extended receptor-binding domain (ExRBD), and S2 core (CR) domain (rSA11/NSP3-fS1, -fNTD, -fRBD, -fExRBD, and -fCR, respectively). Generation of rSA11/fS1 required a foreign-sequence insertion of 2.2-kbp, the largest such insertion yet made into the rotavirus genome. Based on isopycnic centrifugation, rSA11 containing S sequences were denser than wildtype virus, confirming the capacity of the rotavirus to accommodate larger genomes. Immunoblotting showed that rSA11/-fNTD, -fRBD, -fExRBD, and -fCR viruses expressed S products of expected size, with fExRBD expressed at highest levels. These rSA11 viruses were genetically stable during serial passage. In contrast, rSA11/NSP3-fS1 failed to express its expected 80-kDa fS1 product, for unexplained reasons. Moreover, rSA11/NSP3-fS1 was genetically unstable, with variants lacking the S1 insertion appearing during serial passage. Nonetheless, these results emphasize the potential usefulness of rotavirus vaccines as expression vectors of portions of the SARS-CoV-2 S protein (e.g., NTD, RBD, ExRBD, and CR) with sizes smaller than the S1 fragment.

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Production and Application of Multicistronic Constructs for Various Human Disease Therapies

Cited by 32 publications

References 100 publications

Rotavirus as an Expression Platform of Domains of the SARS-CoV-2 Spike Protein

Rotavirus as an Expression Platform of Domains of the SARS-CoV-2 Spike Protein

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

Rotavirus as an Expression Platform of the SARS-CoV-2 Spike Protein

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