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US 2026/0258405 A1Published application
CAPLESS AND TAILLESS THERAPEUTIC EXOGENOUS mRNA AND METHOD TO PRODUCE THE SAME
AssigneeUniversity of South Alabama
InventorsViktor Solodushko, Brian Fouty
Life of this patent
Estimating the term…
Abstract
A biologically functional polynucleotide in the form of therapeutic exogenous mRNA having hairpins in lieu of a cap and poly(A) tail and method of producing the same is provided. The design of a biologically functional polynucleotide allows for a one step method of production. The target mRNA vector or chemically synthesized mRNA molecule of the biologically functional polynucleotide includes internal translation initiation site and delivered gene sequence. Additionally, the target mRNA vector is designed to form stable hairpins at both the 5′ end and 3′ end, wherein said hairpins comprise at least four paired nucleotides in the stem and linked to the closest end with no more than two unpaired nucleotides.
Claims132
A biologically functional polynucleotide comprising,
- a mRNA transcript having a first end and a second end and lacking a cap and a poly(A) tail,
- wherein a first terminal hairpin structure is located at said first end of said mRNA transcript,
- wherein a second terminal hairpin structure is located at said second end of said mRNA transcript,
- wherein said first terminal hairpin structure and said second terminal hairpin structure is a nucleotide sequence configured to stably fold onto itself via nucleotide pairing, and
- wherein said first terminal hairpin structure and said second terminal hairpin structure are linked to said first end and said second end via no more than two unpaired nucleotides,
- wherein said first terminal hairpin structure has a number of individual hairpins that is at least equal to a number of individual hairpins of said second terminal hairpin structure,
- wherein said number of individual hairpins of said first terminal hairpin structure determines an amount of product when said mRNA transcript undergoes translation.
4 more elements
A biologically functional polynucleotide comprising,
- a chemically synthesized mRNA molecule having a first end and a second end and lacking a cap and a poly(A) tail,
- wherein a first terminal hairpin structure is located at said first end of said chemically synthesized mRNA molecule,
- wherein a second terminal hairpin structure is located at said second end of said chemically synthesized mRNA molecule,
- wherein said first terminal hairpin structure and said second terminal hairpin structure are nucleotide sequences configured to stably fold onto themselves via nucleotide pairing, and
- wherein said first terminal hairpin structure and said second terminal hairpin structure are linked to said first end and said second end via no more than two unpaired nucleotides,
- wherein said first terminal hairpin structure has a number of individual hairpins that is at least equal to a number of individual hairpins of said second terminal hairpin structure,
- wherein said number of individual hairpins of said first terminal hairpin structure determines an amount of product when said chemically synthesized mRNA molecule undergoes translation.
4 more elements
A biologically functional polynucleotide comprising,
- a mRNA transcript having a 5′ end and a 3′ end and lacks a poly(A) tail,
- wherein a first terminal hairpin structure is located at said 3′ end of said mRNA transcript,
- wherein said first terminal hairpin structure is a nucleotide sequence configured to stably fold onto itself via nucleotide pairing,
- wherein a number of individual hairpins of said first terminal hairpin structure determines an amount of product when said mRNA transcript undergoes translation,
- wherein said first terminal hairpin structure is linked to said 3′ end via no more than two unpaired nucleotides, and
- a cap operably linked to said 5′ end of said mRNA transcript.
3 more elements
A biologically functional polynucleotide comprising,
- a chemically synthesized mRNA molecule having a 5′ end and a 3′ end and lacks a poly(A) tail,
- wherein a first terminal hairpin structure is located at said 3′ end of said chemically synthesized mRNA molecule,
- wherein said first terminal hairpin structure is a nucleotide sequence configured to stably fold onto itself via nucleotide pairing,
- wherein a number of individual hairpins of said first terminal hairpin structure determines an amount of product when said chemically synthesized mRNA molecule undergoes translation,
- wherein said first terminal hairpin structure is linked to said 3′ end via no more than two unpaired nucleotides, and
- a cap operably linked to said 5′ end of said chemically synthesized mRNA molecule.
3 more elements
A biologically functional polynucleotide comprising,
- a mRNA transcript having a 5′ end and a 3′ end and lacking a cap,
- wherein a first terminal hairpin structure is located at said 5′ end of said mRNA transcript,
- wherein a poly(A) tail sequence is located at said 3′ end of said mRNA transcript,
- wherein a number of individual hairpins of said first terminal hairpin structure determines an amount of product when said mRNA transcript undergoes translation,
- wherein said first terminal hairpin structure is a nucleotide sequence configured to stably fold onto itself via nucleotide pairing, and
- wherein said first terminal hairpin structure is linked to said 5′ end via no more than two unpaired nucleotides.
3 more elements
A biologically functional polynucleotide comprising,
- a chemically synthesized mRNA molecule having a 5′ end and a 3′ end and lacking a cap,
- wherein a first terminal hairpin structure is located at said 5′ end of said chemically synthesized mRNA molecule,
- wherein a poly(A) tail sequence is located at said 3′ end of said chemically synthesized mRNA molecule,
- wherein a number of individual hairpins of said first terminal hairpin structure determines an amount of product when said chemically synthesized mRNA molecule undergoes translation,
- wherein said first terminal hairpin structure is a nucleotide sequence configured to stably fold onto itself via nucleotide pairing, and
- wherein said first terminal hairpin structure is linked to said 5′ end via no more than two unpaired nucleotides.
3 more elements
A DNA template configured to encode a target RNA transcript comprising,
- a DNA source configured to create a target mRNA transcript,
- wherein said target mRNA transcript comprises a first end and a second end and lacks a cap and a poly(A) tail,
- wherein a first terminal hairpin structure is located at said first end of said target mRNA transcript,
- wherein a second terminal hairpin structure is located at said second end of said target mRNA transcript,
- wherein said first terminal hairpin structure and said second terminal hairpin structure are nucleotide sequences configured to stably fold onto themselves via nucleotide pairing, and
- wherein said first terminal hairpin structure and said second terminal hairpin structure are linked to said first end and said second end via no more than two unpaired nucleotides,
- wherein said first terminal hairpin structure has a number of individual hairpins that is at least equal to a number of individual hairpins of said second terminal hairpin structure,
- wherein said number of individual hairpins of said first terminal hairpin structure determines an amount of product when said mRNA transcript undergoes translation.
5 more elements
A DNA template configured to encode a target RNA transcript comprising,
- a DNA source configured to create a target mRNA transcript,
- wherein said target mRNA transcript comprises a first end and a second end and lacks a cap,
- wherein a first terminal hairpin structure is located at said first end of said target mRNA transcript,
- wherein a poly(A) tail is located at said second end of said target mRNA transcript,
- wherein a number of individual hairpins of said first terminal hairpin structure determines an amount of product when said mRNA transcript undergoes translation,
- wherein said first terminal hairpin structure is a nucleotide sequence configured to stably fold onto themselves via nucleotide pairing, and
- wherein said first terminal hairpin structure is linked to said first end via no more than two unpaired nucleotides.
4 more elements
A DNA template configured to encode a target RNA transcript comprising,
- a DNA source configured to create a target mRNA transcript,
- wherein said target mRNA transcript comprises a 5′ end and a 3′ end and lacks a poly(A) tail,
- wherein a first terminal hairpin structure is located at said 3′ end of said target mRNA transcript,
- wherein a cap is located at said 5′ end of said target mRNA transcript,
- wherein a number of individual hairpins of said first terminal hairpin structure determines an amount of product when said mRNA transcript undergoes translation,
- wherein said first terminal hairpin structure is a nucleotide sequence configured to stably fold onto themselves via nucleotide pairing, and
- wherein said first terminal hairpin structure is linked to said 3′ end via no more than two unpaired nucleotides.
4 more elements
A method for creating a biologically functional polynucleotide comprising steps of:
- generating a target mRNA vector having an mRNA transcript possessing a first end and a second end and lacking a cap and a poly(A) tail,
- wherein a first terminal hairpin structure is located at said first end of said mRNA transcript,
- wherein a second terminal hairpin structure is located at said second end of said mRNA transcript,
- wherein said first terminal hairpin structure and said second terminal hairpin structure are nucleotide sequences configured to stably fold onto themselves via nucleotide pairing, and
- wherein said first terminal hairpin structure and said second terminal hairpin structure are linked to said first end and said second end via no more than two unpaired nucleotides,
- wherein said target mRNA vector has one of a mRNA transcript and a chemically synthesized mRNA molecule,
- wherein said first terminal hairpin structure has a number of individual hairpins that is at least equal to a number of individual hairpins of said second terminal hairpin structure,
- wherein said number of individual hairpins of said first terminal hairpin structure determines an amount of a target mRNA vector product,
- inserting said target mRNA vector sequence into a DNA source to create a DNA template,
- performing in vitro transcription on said DNA template using DNA-dependent RNA polymerase to obtain a target mRNA vector product, and
- purifying said target mRNA vector product to produce therapeutic exogenous mRNA.
8 more elements
A method for creating a biologically functional polynucleotide comprising steps of:
- generating a target mRNA vector having a chemically synthesized mRNA molecule possessing a first end and a second end and lacking a cap and a poly(A) tail,
- wherein a first terminal hairpin structure is located at said first end of said chemically synthesized mRNA molecule,
- wherein a second terminal hairpin structure is located at said second end of said chemically synthesized mRNA molecule,
- wherein said first terminal hairpin structure and said second terminal hairpin structure are nucleotide sequences configured to stably fold onto themselves via nucleotide pairing, and
- wherein said first terminal hairpin structure and said second terminal hairpin structure are linked to said first end and said second end via no more than two unpaired nucleotides,
- wherein said first terminal hairpin structure has a number of individual hairpins that is at least equal to a number of individual hairpins of said second terminal hairpin structure,
- wherein said number of individual hairpins of said first terminal hairpin structure determines an amount of said target mRNA vector,
- purifying said target mRNA vector to produce therapeutic exogenous mRNA.
5 more elements
A method for creating a biologically functional polynucleotide comprising steps of:
- generating a target mRNA vector having an mRNA transcript possessing a 5′ end and a 3′ end and lacks a poly(A) tail,
- wherein a first terminal hairpin structure is located at said 3′ end of said mRNA transcript,
- wherein a cap is located at said 5′ end of said mRNA transcript,
- wherein said first terminal hairpin structure is a nucleotide sequence configured to stably fold onto itself via nucleotide pairing,
- wherein a number of individual hairpins of said first terminal hairpin structure determines an amount of a target mRNA vector product,
- wherein said first terminal hairpin structure is linked to said 3′ end via no more than two unpaired nucleotides,
- inserting said target mRNA vector sequence into a DNA source to create a DNA template,
- performing in vitro transcription on said DNA template using DNA-dependent RNA polymerase to obtain a said target mRNA vector product, and
- purifying said target mRNA vector product to produce therapeutic exogenous mRNA.
6 more elements
A method for creating a biologically functional polynucleotide comprising steps of:
- generating a target mRNA vector having an mRNA transcript possessing a 5′ end and a 3′ end and lacking a cap,
- wherein a first terminal hairpin structure is located at said 5′ end of said mRNA transcript,
- wherein a poly(A) tail is located at said 3′ end of said mRNA transcript,
- wherein said first terminal hairpin structure is a nucleotide sequence configured to stably fold onto itself via nucleotide pairing,
- wherein a number of individual hairpins of said first terminal hairpin structure determines an amount of a target mRNA vector product,
- wherein said first terminal hairpin structure is linked to said 5′ end via no more than two unpaired nucleotides,
- inserting said target mRNA vector into a DNA source to create a DNA template,
- performing in vitro transcription on said DNA template using DNA-dependent RNA polymerase to obtain a target mRNA vector product, and
- purifying said target mRNA vector product to produce therapeutic exogenous mRNA.
6 more elements
A method for creating a biologically functional polynucleotide comprising steps of:
- generating a target mRNA vector having a chemically synthesized mRNA molecule possessing a 5′ end and a 3′ end and lacks a poly(A) tail,
- wherein a first terminal hairpin structure is located at said 3′ end of said chemically synthesized mRNA molecule,
- wherein a cap is located at said 5′ end of said chemically synthesized mRNA molecule,
- wherein said first terminal hairpin structure is a nucleotide sequence configured to stably fold onto itself via nucleotide pairing,
- wherein a number of individual hairpins of said first terminal hairpin structure determines an amount of a target mRNA vector product,
- wherein said first terminal hairpin structure is linked to said 3′ end via no more than two unpaired nucleotides,
- obtaining said target mRNA vector product using said target mRNA vector, and
- purifying said target mRNA vector product to produce therapeutic exogenous mRNA.
5 more elements
A method for creating a biologically functional polynucleotide comprising steps of:
- generating a target mRNA vector having a chemically synthesized mRNA molecule possessing a 5′ end and a 3′ end and lacking a cap,
- wherein a first terminal hairpin structure is located at said 5′ end of said chemically synthesized mRNA molecule,
- wherein a poly(A) tail is located at said 3′ end of said chemically synthesized mRNA molecule,
- wherein said first terminal hairpin structure is a nucleotide sequence configured to stably fold onto itself via nucleotide pairing,
- wherein a number of individual hairpins of said first terminal hairpin structure determines an amount of target mRNA vector product,
- wherein said first terminal hairpin structure is linked to said 5′ end via no more than two unpaired nucleotides,
- obtaining a target mRNA vector product using said target mRNA vector, and
- purifying said target mRNA vector product to produce therapeutic exogenous mRNA.
5 more elements
A product for use as a therapeutic agent, wherein said product is prepared by:
- inserting a target mRNA vector sequence into a DNA source to create a DNA template,
- wherein said target mRNA vector sequence encodes a first terminal hairpin structure that is located at a first end of a target mRNA vector and a second terminal hairpin structure that is located at a second end of said target mRNA vector,
- wherein an internal translation initiation site is incorporated into said target mRNA vector,
- wherein said target mRNA vector sequence lacks a cap and a poly(A) tail,
- wherein said first terminal hairpin structure has a number of individual hairpins that is at least equal to a number of individual hairpins of said second terminal hairpin structure,
- wherein said number of individual hairpins of said first terminal hairpin structure determines an amount of target mRNA vector product,
- performing in vitro transcription on said DNA template using DNA-dependent RNA polymerase to obtain a target mRNA vector product, and
- purifying said target mRNA vector product to produce a therapeutic agent,
- wherein said therapeutic agent is said product.
6 more elements
A product for use as a therapeutic agent, wherein said product is prepared by:
- inserting a target mRNA vector sequence into a DNA source to create a DNA template,
- wherein said target mRNA vector sequence encodes a first terminal hairpin structure onto a 5′ end of a target mRNA vector and a poly(A) tail at a 3′ end of said target mRNA vector,
- wherein said target mRNA vector sequence lacks a cap,
- wherein a number of individual hairpins of said first terminal hairpin structure determines an amount of a target mRNA vector product,
- wherein an internal translation initiation site is incorporated into said target mRNA vector,
- performing in vitro transcription on said DNA template using DNA-dependent RNA polymerase to obtain a target mRNA vector product, and
- purifying said target mRNA vector product to produce a therapeutic agent,
- wherein said therapeutic agent is said product.
5 more elements
A product for use as a therapeutic agent, wherein said product is prepared by:
- inserting a target mRNA vector sequence into a DNA source to create a DNA template,
- wherein said target mRNA vector sequence encodes a first terminal hairpin structure onto a 3′ end of a target mRNA vector and a cap at a 5′ end of said target mRNA vector,
- wherein an internal translation initiation site is incorporated into said target mRNA vector,
- wherein said target mRNA vector sequence lacks a poly(A) tail,
- wherein a number of individual hairpins of said first terminal hairpin structure determines an amount of a target mRNA vector product,
- performing in vitro transcription on said DNA template using DNA-dependent RNA polymerase to obtain a target mRNA vector product, and
- performing in vitro transcription on said DNA template using DNA-dependent RNA polymerase to obtain a target mRNA vector product, and
- purifying said target mRNA vector product to produce a therapeutic agent,
- wherein said therapeutic agent is said product.
6 more elements
A product for use as a therapeutic agent, wherein said product is prepared by:
- performing chemical synthesis on an mRNA vector having a target mRNA vector sequence to create a chemically synthesized mRNA molecule,
- wherein said mRNA vector has a first end and a second end,
- wherein said target mRNA vector sequence encodes a first terminal hairpin structure onto a first end of a target mRNA vector and a second terminal hairpin structure onto a second end of said target mRNA vector,
- wherein an internal translation initiation site is incorporated into said chemically synthesized mRNA molecule,
- wherein said target mRNA vector sequence lacks a cap and a poly(A) tail,
- wherein said first terminal hairpin structure has a number of individual hairpins that is at least equal to a number of individual hairpins of said second terminal hairpin structure,
- wherein said number of individual hairpins of said first terminal hairpin structure determines an amount of a target mRNA vector product,
- performing transcription on said chemically synthesized mRNA molecule to obtain a target mRNA vector product,
- purifying said target mRNA vector product to produce a therapeutic agent,
- wherein said therapeutic agent is said product.
7 more elements
A product for use as a therapeutic agent, wherein said product is prepared by:
- performing chemical synthesis on an mRNA vector having a target mRNA vector sequence to create a chemically synthesized mRNA molecule,
- wherein said target mRNA vector sequence encodes a first terminal hairpin structure at a 5′ end of a target mRNA vector and a poly(A) tail at a 3′ end of said target mRNA vector,
- wherein said target mRNA vector sequence lacks a cap,
- wherein a number of individual hairpins of said first terminal hairpin structure determines an amount of a target mRNA vector product,
- wherein an internal translation initiation site is incorporated into said chemically synthesized mRNA molecule,
- performing transcription on said chemically synthesized mRNA molecule to obtain a target mRNA vector product,
- purifying said target mRNA vector product to produce a therapeutic agent,
- wherein said therapeutic agent is said product
5 more elements
A product for use as a therapeutic agent, wherein said product is prepared by:
- performing chemical synthesis on an mRNA vector having a target mRNA vector sequence to create a chemically synthesized mRNA molecule,
- wherein said target mRNA vector sequence encodes a first terminal hairpin structure at a 3′ end of a target mRNA vector and a cap at a 5′ end of said target mRNA vector,
- wherein an internal translation initiation site is incorporated into said chemically synthesized mRNA molecule,
- wherein said target mRNA vector sequence lacks a poly(A) tail,
- wherein a number of individual hairpins of said first terminal hairpin structure determines an amount of a target mRNA vector product,
- performing transcription on said chemically synthesized mRNA molecule to obtain a target mRNA vector product,
- purifying said target mRNA vector product to produce a therapeutic agent,
- wherein said therapeutic agent is said product.
5 more elements
Dependencies are inferred from the claim text.