HIV pre-immunization and immunotherapy
Inventors
Pauza, Charles David • Li, Haishan • Lahusen, Tyler • Galvin, Jeff
Assignees
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Publication Number
US-11612649-B2
Publication Date
2023-03-28
Expiration Date
Abstract
The present invention relates generally to immunization and immunotherapy for the treatment or prevention of HIV. In particular, the methods include in vivo and/or ex vivo enrichment of HIV-specific CD4+ T cells.
Core Innovation
The document relates to treating cells infected with HIV by obtaining peripheral blood mononuclear cells (PBMC) from an HIV-infected subject, contacting the PBMC ex vivo with a therapeutically effective amount of a second stimulatory agent, and transducing the PBMC ex vivo with a viral delivery system encoding defined genetic elements. The subject is previously immunized with a therapeutically effective amount of a first stimulatory agent, and the transduced PBMC are cultured for at least 1 day.
The viral delivery system encodes genetic elements defined by sequence identity relationships to specific SEQ ID NOs, including at least two of sequences comprising at least 80% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 108, or each of sequences comprising at least 80% sequence identity with SEQ ID NO: 97, SEQ ID NO: 6, and SEQ ID NO: 7. Additional embodiments describe lentiviral vectors and lentiviral particles that include envelope proteins capable of infecting target cells together with encoded microRNA clusters requiring at least 90% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 108.
The encoded microRNA cluster and related small RNA or microRNA/shRNA cargos are used to reduce CCR5 expression and suppress or impair HIV gene expression and replication by targeting HIV RNA regions such as Tat and Vif. The document also reports inhibition of CCR5-tropic HIV replication and reductions in CCR5, Tat mRNA, and Vif protein for lead candidates and combined clusters.
Claims Coverage
The independent claims cover ex vivo PBMC treatment workflows with prior immunization, ex vivo stimulation, lentiviral transduction, and culturing; lentiviral vectors and lentiviral particles encoding microRNA clusters defined by sequence-identity requirements; and modified primary T cells infected with the defined lentiviral particle architectures. Across these claims, the genetic element payload is restricted using explicit sequence-identity thresholds to specified SEQ ID NOs, and the treatment context is directed to HIV infection.
Ex vivo PBMC treatment after prior immunization with defined sequence-identity genetic elements
Obtaining, or having obtained, PBMC from a subject infected with HIV, wherein the subject has been previously immunized with a therapeutically effective amount of a first stimulatory agent; contacting, or having contacted, the PBMC with a therapeutically effective amount of a second stimulatory agent, wherein the contacting is carried out ex vivo; transducing, or having transduced, the PBMC ex vivo with a viral delivery system encoding at least one genetic element, wherein at least one encoded genetic element comprises each of: a sequence comprising at least 80% sequence identity with SEQ ID NO: 2 and a sequence comprising at least 80% sequence identity with SEQ ID NO: 108; or two of: a sequence comprising at least 80% sequence identity with SEQ ID NO: 97, a sequence comprising at least 80% sequence identity with SEQ ID NO: 6, and a sequence comprising at least 80% sequence identity with SEQ ID NO: 7; and culturing, or having cultured, the transduced PBMC for at least 1 day.
Lentiviral treatment of HIV infection with ex vivo leukocyte and PBMC handling
Removing, or having removed, leukocytes from the subject, wherein the subject has been previously immunized with a therapeutically effective amount of a first stimulatory agent; purifying, or having purified, PBMC ex vivo from the leukocytes; contacting, or having contacted, the PBMC ex vivo with a therapeutically effective amount of a second stimulatory agent; transducing, or having transduced, the PBMC ex vivo with a viral delivery system encoding at least one genetic element, wherein the at least one genetic element comprises each of: a sequence comprising at least 80% sequence identity with SEQ ID NO: 2 and a sequence comprising at least 80% sequence identity with SEQ ID NO: 108; or two of: a sequence comprising at least 80% sequence identity with SEQ ID NO: 97, a sequence comprising at least 80% sequence identity with SEQ ID NO: 6, and a sequence comprising at least 80% sequence identity with SEQ ID NO: 7; and culturing, or having cultured, the transduced PBMC for at least 1 day.
Lentiviral vector with encoded microRNA cluster defined by sequence identity
A lentiviral vector comprising an encoded microRNA cluster, wherein the encoded microRNA cluster comprises a sequence comprising at least 90% sequence identity with SEQ ID NO: 1, at least 90% sequence identity with SEQ ID NO: 2, and at least 90% sequence identity with SEQ ID NO: 108.
Lentiviral particle for infecting a target cell with encoded microRNA cluster
A lentiviral particle capable of infecting a target cell, the lentiviral particle comprising an envelope protein capable of infecting the target cell and an encoded microRNA cluster, wherein the encoded microRNA cluster comprises a sequence comprising at least 90% sequence identity with SEQ ID NO: 1, at least 90% sequence identity with SEQ ID NO: 2, and at least 90% sequence identity with SEQ ID NO: 108.
Modified primary T cell infected with defined lentiviral particle microRNA cluster architecture
A modified cell comprising a primary T cell infected with a lentiviral particle, wherein the lentiviral particle comprises an envelope protein capable of infecting the target cell and an encoded microRNA cluster, wherein the encoded microRNA cluster comprises a sequence comprising at least 90% sequence identity with SEQ ID NO: 1, at least 90% sequence identity with SEQ ID NO: 2, and at least 90% sequence identity with SEQ ID NO: 108.
Ex vivo contacting and lentiviral particle transduction of HIV-infected PBMC
Contacting peripheral blood mononuclear cells (PBMC) isolated from a subject infected with HIV with a therapeutically effective amount of an ex vivo stimulatory agent, wherein the contacting is conducted ex vivo; transducing the PBMC ex vivo with a lentiviral particle comprising an envelope protein capable of infecting the PBMC and an encoded microRNA cluster, wherein the encoded microRNA cluster comprises a sequence comprising at least 90% sequence identity with SEQ ID NO: 1, at least 90% sequence identity with SEQ ID NO: 2, and at least 90% sequence identity with SEQ ID NO: 108.
Lentiviral vector encoded genetic element payload
A lentiviral vector comprising at least one encoded genetic element, wherein the at least one encoded genetic element comprises at least two of: a sequence comprising at least 80% sequence identity with SEQ ID NO: 1, a sequence comprising at least 80% sequence identity with SEQ ID NO: 2, and a sequence comprising at least 80% sequence identity with SEQ ID NO: 108; or each of: a sequence comprising at least 80% sequence identity with SEQ ID NO: 97, a sequence comprising at least 80% sequence identity with SEQ ID NO: 6, and a sequence comprising at least 80% sequence identity with SEQ ID NO: 7.
Ex vivo PBMC treatment with viral delivery system encoding alternative SEQ ID defined genetic elements
Obtaining, or having obtained, peripheral blood mononuclear cells (PBMC) from a subject infected with HIV, wherein the subject has been previously immunized with a therapeutically effective amount of a first stimulatory agent; contacting, or having contacted, the PBMC with a therapeutically effective amount of a second stimulatory agent, wherein the contacting is carried out ex vivo; transducing, or having transduced, the PBMC ex vivo with a viral delivery system encoding at least one genetic element, wherein at least one encoded genetic element comprises at least two of: a sequence comprising at least 80% sequence identity with SEQ ID NO: 1, a sequence comprising at least 80% sequence identity with SEQ ID NO: 2, and a sequence comprising at least 80% sequence identity with SEQ ID NO: 108; or each of: a sequence comprising at least 80% sequence identity with SEQ ID NO: 97, a sequence comprising at least 80% sequence identity with SEQ ID NO: 6, and a sequence comprising at least 80% sequence identity with SEQ ID NO: 7; and culturing, or having cultured, the transduced PBMC for at least 1 day.
Overall, claim coverage centers on ex vivo treatment of HIV-infected cells using PBMC that are ex vivo stimulated and lentivirally transduced with genetic elements defined by explicit sequence-identity relationships to specific SEQ ID NOs. Separate independent claims also cover lentiviral vector and lentiviral particle constructs containing encoded microRNA clusters defined by at least 90% identity, and a modified primary T cell infected with such a lentiviral particle.
Stated Advantages
Reduces CCR5 expression by inhibiting production of chemokine receptor CCR5.
Targets HIV RNA and suppresses or impairs HIV gene expression and replication.
Inhibition of CCR5-tropic HIV replication.
Reduction of CCR5 knockdown for lead candidates reported in the partial content.
Reduction of Vif protein as reported in the partial content for miR21Vif.
Reduction of Tat mRNA with higher potency than miR155Tat as reported in the partial content for miR185Tat.
Combined miRNA cluster producing strong reductions of CCR5, Tat, and Vif and associated HIV replication inhibition as reported in the partial content.
Provides a functional cure immunotherapy approach combining in vivo immunization with ex vivo transduction and optional infusion.
Achieving HIV-specific, lentivirus-transduced CD4+ T cells for a “functional cure” approach as reported in the partial content.
Documented Applications
Treating cells infected with HIV using ex vivo PBMC contacting, lentiviral ex vivo transduction encoding sequence-identity-defined genetic elements or encoded microRNA clusters, and culturing transduced PBMC for at least 1 day.
Treating HIV infection in a subject by removing leukocytes, purifying PBMC ex vivo, ex vivo contacting with a second stimulatory agent, ex vivo transducing PBMC with a viral delivery system encoding sequence-identity-defined genetic elements, and culturing the transduced PBMC for at least 1 day.
Ex vivo treatment of HIV-infected cells using PBMC contacted with an ex vivo stimulatory agent and transduced with a lentiviral particle encoding an encoded microRNA cluster defined by sequence identity.
Ex vivo functional proof-of-concept for achieving HIV-specific, lentivirus-transduced CD4+ T cells for a “functional cure” approach.
Inhibition of CCR5-tropic HIV replication using combinations of microRNA clusters targeting CCR5, Tat, and Vif as reported in the partial content.
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