Materials and methods for producing improved lentiviral vector particles
Inventors
Nicolai, Christopher James • Tareen, Semih U.
Assignees
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Publication Number
US-9713635-B2
Publication Date
2017-07-25
Expiration Date
Abstract
Materials and methods useful for generating highly mannosylated pseudotyped lentiviral vector particles comprising a Vpx protein are provided.
Core Innovation
The invention is directed to a composition of pseudotyped lentiviral vector particles that include a Vpx protein or a Vpr protein retaining SAMHD1-inhibiting activity, a lentiviral vector genome comprising an exogenous polynucleotide of interest, and a plurality of envelope glycoproteins that preferentially bind cells expressing DC-SIGN (CD209). The pseudotyped lentiviral vector particles are produced from a virus packaging cell cultured in a culture medium comprising kifunensine.
Compared with control pseudotyped lentiviral vector particles prepared in the absence of kifunensine, the produced pseudotyped lentiviral vector particles are more highly mannosylated. The content states that the kifunensine culture condition increases mannose-rich glycosylation on the envelope glycoproteins and is associated with DC-SIGN/DC-SIGNR targeting and productive DC transduction.
The composition further combines DC-SIGN preferential targeting with SAMHD1 inhibition using Vpx or SAMHD1-inhibiting Vpr. The disclosed embodiments also describe compatibility with lentiviral vector genomes including self-inactivating 3′ LTR and integration-defective/integration-deficient genomes, and report in vivo induction of polyfunctional CD8 T cell responses with antigen-specific protection and anti-tumor effects.
Claims Coverage
The claim set identifies one independent claim covering a DC-SIGN-targeted, Vpx/Vpr-containing pseudotyped lentiviral vector composition whose production uses kifunensine to obtain increased mannosylation compared to kifunensine-free controls. The merged claim coverage contains three inventive features.
SAMHD1-inhibiting Vpx or Vpr in DC-SIGN-targeted pseudotyped lentiviral particles
A composition comprising pseudotyped lentiviral vector particles including a Vpx protein or a Vpr protein that retains SAMHD1-inhibiting activity, and a plurality of envelope glycoproteins that preferentially bind cells expressing DC-SIGN.
Exogenous polynucleotide lentiviral genome
The pseudotyped lentiviral vector particles include a lentiviral vector genome comprising an exogenous polynucleotide of interest.
Kifunensine-mediated production yielding more highly mannosylated particles
The pseudotyped lentiviral vector particles are produced from a virus packaging cell cultured in a culture medium comprising kifunensine, and the pseudotyped lentiviral vector particles are more highly mannosylated than control pseudotyped lentiviral vector particles prepared in the absence of kifunensine.
The core claim coverage combines DC-SIGN-preferential envelope glycoprotein targeting, SAMHD1-inhibiting Vpx or Vpr, and a lentiviral vector genome comprising an exogenous polynucleotide, together with kifunensine-mediated increased mannosylation relative to kifunensine-free controls.
Stated Advantages
Particles are more highly mannosylated compared to control pseudotyped lentiviral vector particles prepared without kifunensine.
DC-SIGN/DC-SIGNR targeting supports productive DC transduction and immune response outcomes as described in the document.
Unexpectedly higher transduction of dendritic cells expressing DC-SIGN when both DC-SIGN targeting and SAMHD1 inhibition are present.
In vivo induction of polyfunctional CD8 T cell responses with antigen-specific protection and anti-tumor effects.
Documented Applications
In vivo delivery of pseudotyped lentiviral vectors to dendritic cells for antigen-specific adoptive immunotherapy, including induction of polyfunctional CD8 T cell responses and antigen-specific protection with anti-tumor effects.
DC-SIGN-targeted delivery using pseudotyped lentiviral vector particles comprising Vpx/Vpr retaining SAMHD1-inhibiting activity and envelope glycoproteins that preferentially bind DC-SIGN, with described use in transduction of DCs expressing DC-SIGN.
In vivo targeting of mouse DCs, including SIGNR1 expression and draining lymph node transduction.
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