Constrained lipids and methods of use thereof

Abstract

This invention details various lipids, compositions, and / or optimized systems and delivery methods for delivering nucleic acid sequences, polypeptides, or peptides for vaccination against infectious agents.

Description

Technical field

[0001] The present invention relates to optimized systems for the delivery of nucleic acid sequences, polypeptides or peptides and methods of using such optimized systems to treat diseases, disorders and / or conditions. prior art

[0002] Proteins have always been the standard of care, but over the past few years, the use of nucleic acids has become increasingly prominent as a therapeutic modality for a variety of diseases and therapeutic indications. Several companies have shown that nucleic acids (e.g., siRNA, mRNA, circular RNA, DNA, ASO, etc.) may be more effective when compared to protein-based therapies, but targeted delivery systems are required for both nucleic acid and protein therapies. To ensure that the therapeutic agent is localized to the targeted cells, tissues or organs.

[0003] Current delivery systems, including lipid-based delivery systems (such as lipid nanoparticles) focus on protecting the cargo being delivered, but not on the lipids used in the delivery system, and often do not Positioned delivery of carriers or delivery systems. There is a need for improved lipid-based delivery systems in this technology. Contents of the invention

[0004] The present invention provides novel lipids useful in delivery vehicles for delivery systems and a tropism discovery platform for screening and developing targeting systems for localized delivery of nucleic acid and protein therapeutics, such as to immune cells.

[0005] In one aspect of the invention, provided herein is a lipid having any one of Formulas I-VII or a pharmaceutically acceptable salt or solvate thereof, or any lipid in Table (I) Substances or salts or solvates thereof, see below, are collectively referred to as "lipids of the present invention (Lipids of the Disclosure)" and each is individually referred to as "lipid of the present invention (Lipid of the Disclosure)".

[0006] In one aspect of the invention, this article provides a pharmaceutical composition comprising:

[0007] a) a polynucleotide encoding at least one protein of interest, and

[0008] b) Delivery vehicle containing at least one lipid

[0009] Wherein the composition triggers an immune response in the individual.

[0010] In one aspect, the polynucleotide is DNA.

[0011] In one aspect, the polynucleotide is RNA.

[0012] In one aspect, the RNA is short interfering RNA (siRNA).

[0013] In one aspect, siRNA inhibits or inhibits the expression of a target of interest in a cell.

[0014] In one aspect, inhibition or inhibition is about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20- 40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30- 60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40- 90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60- 80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80- 100%, 90-95%, 90-100% or 95-100%.

[0015] In one aspect, the polynucleotide is substantially cyclic.

[0016] In one aspect, the polynucleotide includes an internal ribosome entry site (IRES) sequence operably linked to the payload sequence region.

[0017] In one aspect, the IRES sequence includes a sequence derived from picornavirus complementary DNA, encephalomyocarditis virus (EMCV) complementary DNA, poliovirus complementary DNA, or an antennapedia gene from Drosophila melanogaster.

[0018] In one aspect, the polynucleotide includes a termination element, wherein the termination element includes at least one termination codon.

[0019] In one aspect, the polynucleotide includes regulatory elements.

[0020] In one aspect, the polynucleotide includes at least one masking agent.

[0021] In one aspect, in vitro transcription is used to generate substantially circular polynucleotides.

[0022] In one aspect, the payload sequence region includes non-coding nucleic acid sequences.

[0023] In one aspect, the payload sequence region includes a coding nucleic acid sequence.

[0024] In one aspect, the coding nucleic acid sequence encodes a Campylobacter jejuni protein of interest. In one aspect, the coding nucleic acid sequence encodes a Clostridium difficile protein of interest. In one aspect, the encoding nucleic acid sequence encodes a protein of interest in Entamoeba histolytica. In one aspect, the encoding nucleic acid sequence encodes an enterotoxin B protein of interest. In one aspect, the encoding nucleic acid sequence encodes a Norwalk virus or norovirus protein of interest. In one aspect, the encoding nucleic acid sequence encodes a protein of interest from Helicobacter pylori. In one aspect, the encoding nucleic acid sequence encodes a rotavirus protein of interest. In one aspect, the coding nucleic acid sequence encodes a protein of interest in candida yeast. In one aspect, the encoding nucleic acid sequence encodes a protein of interest for a coronavirus. In one aspect, the encoding nucleic acid sequence encodes a SARS-CoV protein of interest. In one aspect, the encoding nucleic acid sequence encodes a SARS-CoV-2 protein of interest. In one aspect, the encoding nucleic acid sequence encodes a protein of interest for MERS-CoV. In one aspect, the encoding nucleic acid sequence encodes an enterovirus 71 protein of interest. In one aspect, the encoding nucleic acid sequence encodes a protein of interest for Epstein-Barr virus. In one aspect, the encoding nucleic acid sequence encodes a protein of interest in a Gram-negative bacterium. In one aspect, the Gram-negative bacterium is Bordetella. In one aspect, the encoding nucleic acid sequence encodes a protein of interest in a Gram-positive bacterium. In one aspect, the Gram-positive bacterium is Clostridium tetani. In one aspect, the Gram-positive bacterium is Francisella tularensis. In one aspect, the Gram-positive bacteria are bacteria of the genus Streptococcus. In one aspect, the Gram-positive bacteria are Staphylococcus bacteria. In one aspect, the encoding nucleic acid sequence encodes a protein of hepatitis concern. In one aspect, the encoding nucleic acid sequence encodes a protein of interest for Human Cytomegalovirus. In one aspect, the encoding nucleic acid sequence encodes a human immunodeficiency virus protein of interest. In one aspect, the encoding nucleic acid sequence encodes a human papilloma virus protein of interest. In one aspect, the encoding nucleic acid sequence encodes an influenza protein of interest. In one aspect, the encoding nucleic acid sequence encodes a protein of interest for John Cunningham Virus. In one aspect, the encoding nucleic acid sequence encodes a Mycobacterium protein of interest. In one aspect, the encoding nucleic acid sequence encodes a protein of interest for Poxviruses. In one aspect, the encoding nucleic acid sequence encodes a Pseudomonas aeruginosa protein of interest. In one aspect, the coding nucleic acid sequence encodes a protein of interest for Respiratory Syncytial Virus. In one aspect, the encoding nucleic acid sequence encodes a protein of interest for Rubella virus. In one aspect, the encoding nucleic acid sequence encodes a protein of interest of Varicella zoster virus. In one aspect, the encoding nucleic acid sequence encodes a protein of interest for Chikungunya virus. In one aspect, the encoding nucleic acid sequence encodes a protein of interest for a Dengue virus. In one aspect, the encoding nucleic acid sequence encodes a protein of interest for Rabies virus. In one aspect, the coding nucleic acid sequence encodes a protein of interest for Trypanosoma cruzi and / or Chagas disease. In one aspect, the encoding nucleic acid sequence encodes a protein of interest for Ebola virus. In one aspect, the coding nucleic acid sequence encodes a protein of interest in Plasmodium falciparum. In one aspect, the encoding nucleic acid sequence encodes a protein of interest for Marburg virus. In one aspect, the encoding nucleic acid sequence encodes a protein of interest for Japanese encephalitis virus. In one aspect, the encoding nucleic acid sequence encodes a protein of interest for St. Louis encephalitis virus. In one aspect, the encoding nucleic acid sequence encodes a West Nile Virus protein of interest. In one aspect, the encoding nucleic acid sequence encodes a protein of interest for Yellow Fever virus. In one aspect, the encoding nucleic acid sequence encodes a protein of interest from Bacillus anthracis. In one aspect, the encoding nucleic acid sequence encodes a protein of interest for Botulinum toxin. In one aspect, the encoding nucleic acid sequence encodes a Ricin protein of interest. In one aspect, the coding nucleic acid sequence encodes a Shiga toxin and / or a Shiga-like toxin protein of interest.

[0025] In one aspect, the polynucleotide contains at least one modification.

[0026] In one aspect, at least 20% of the bases are modified. In one aspect, at least 30% of the bases are modified. In one aspect, at least 40% of the bases are modified. In one aspect, at least 50% of the bases are modified. In one aspect, at least 60% of the bases are modified. In one aspect, at least 70% of the bases are modified. In one aspect, at least 80% of the bases are modified. In one aspect, at least 90% of the bases are modified. In one aspect, at least 100% of the bases are modified. In one aspect, a particular base contains at least one modification.

[0027] In one aspect, the base is adenine. In one aspect, at least 20% of the adenine bases are modified. In one aspect, at least 30% of the adenine bases are modified. In one aspect, at least 40% of the adenine bases are modified. In one aspect, at least 50% of the adenine bases are modified. In one aspect, at least 60% of the adenine bases are modified. In one aspect, at least 70% of the adenine bases are modified. In one aspect, at least 80% of the adenine bases are modified. In one aspect, at least 90% of the adenine bases are modified. In one aspect, at least 100% of the adenine bases are modified.

[0028] In one aspect, the base is guanine. In one aspect, at least 20% of the guanine bases are modified. In one aspect, at least 30% of the guanine bases are modified. In one aspect, at least 40% of the guanine bases are modified. In one aspect, at least 50% of the guanine bases are modified. In one aspect, at least 60% of the guanine bases are modified. In one aspect, at least 70% of the guanine bases are modified. In one aspect, at least 80% of the guanine bases are modified. In one aspect, at least 90% of the guanine bases are modified. In one aspect, at least 100% of the guanine bases are modified.

[0029] In one aspect, the base is cytosine. In one aspect, at least 20% of the cytosine bases are modified. In one aspect, at least 30% of the cytosine bases are modified. In one aspect, at least 40% of the cytosine bases are modified. In one aspect, at least 50% of the cytosine bases are modified. In one aspect, at least 60% of the cytosine bases are modified. In one aspect, at least 70% of the cytosine bases are modified. In one aspect, at least 80% of the cytosine bases are modified. In one aspect, at least 90% of the cytosine bases are modified. In one aspect, at least 100% of the cytosine bases are modified.

[0030] In one aspect, the base is uracil. In one aspect, at least 20% of the uracil bases are modified. In one aspect, at least 30% of the uracil bases are modified. In one aspect, at least 40% of the uracil bases are modified. In one aspect, at least 50% of the uracil bases are modified. In one aspect, at least 60% of the uracil bases are modified. In one aspect, at least 70% of the uracil bases are modified. In one aspect, at least 80% of the uracil bases are modified. In one aspect, at least 90% of the uracil bases are modified. In one aspect, at least 100% of the uracil bases are modified.

[0031] In one aspect, the at least one modification is pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4- Thio-pseudouridine, 2-Thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5- Proparnyl-uridine, 1-propynyl-pseudouridine, 5-taurinemethyluridine, 1-taurinemethyl-pseudouridine, 5-taurinemethyl-2-sulfide Methyl-uridine, 1-taurinemethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine Glycoside, 2-thio-1-methyl-pseudouridine, 1-methyl-1-desa-pseudouridine, 2-thio-1-methyl-1-desa-pseudouridine, di Hydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudine, 2-methoxyuridine, 2-methoxy-4-thio- Uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- Pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine , 4-Thio-1-methyl-1-desa-pseudocytidine, 1-methyl-1-desa-pseudocytidine, zebularine, 5-aza-ze Zebraline, 5-methyl-zebraline, 5-aza-2-thio-zebraline, 2-thio-zebraline, 2-methoxy-cytidine, 2- Methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6- Diaminopurine, 7-desa-adenine, 7-desa-8-aza-adenine, 7-desa-2-aminopurine, 7-desa-8-aza-2-amine Purine, 7-desa-2,6-diaminopurine, 7-desa-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-prenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinol Aminomethyl adenosine, N6-threonylamine methyl adenosine, 2-Methylthio-N6-hydroxybutylamine methyl adenosine, N6,N6-dimethyladenosine , 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wyosine ( wybutosine), 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-de Nitrogen-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methyl Guanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-side oxy-guanosine, 7-methyl-8-side oxy-guanosine, 1-methyl-6- Thio-guanosine, N2-methyl-6-thio-guanosine or N2,N2-dimethyl-6-thio-guanosine.

[0032] In one aspect, the pharmaceutical composition includes at least one cationic lipid selected from the group consisting of: any lipid in Table (I), any lipid having a structure of Formulas I-VII, and combinations thereof.

[0033] In one aspect, the pharmaceutical composition includes additional cationic lipids.

[0034] In one aspect, the pharmaceutical composition includes neutral lipids.

[0035] In one aspect, the pharmaceutical composition includes anionic lipids.

[0036] In one aspect, the pharmaceutical composition includes an auxiliary lipid.

[0037] In one aspect, the pharmaceutical composition includes stealth lipids.

[0038] In one aspect, the weight ratio of lipid to polynucleotide is from about 100:1 to about 1:1.

[0039] In one aspect, the pharmaceutical composition delivers a carrier or payload to the immune cells of an individual in need thereof. The immune cells can be T cells, such as CD8+ T cells, CD4+ T cells or T regulatory cells. Immune cells may also be macrophages or dendritic cells, for example.

[0040] In one aspect, the vaccine formulation includes a pharmaceutical composition.

[0041] In one aspect, the vaccine is prepared having any one of Formulas (I)-(VII).

[0042] In one aspect, provided herein is a method of vaccinating an individual against an infectious agent, comprising contacting the individual with a vaccine formulation or preparation and eliciting an immune response.

[0043] In one aspect, the infectious agent is Campylobacter jejuni, Clostridium difficile, Entamoeba dysenteriae, Enterotoxin B, Norwalk or Norovirus, Helicobacter pylori, Rotavirus, Candida yeast, Coronavirus (including SARS-CoV, SARS-CoV-2 and MERS-CoV), enterovirus 71, Epstein-Barr virus, Gram-negative bacteria (including Bordetella), Gram-positive bacteria (including Tetanus Clostridium tularensis, Francisella tularensis, Streptococcus bacteria and Staphylococcus bacteria), and hepatitis, human cytomegalovirus, human immunodeficiency virus, human papilloma virus, influenza, John Cunningham virus, mycobacteria , poxvirus, Pseudomonas aeruginosa, respiratory syncytial virus, German measles virus, varicella-zoster virus, trichomoniasis virus, dengue virus, rabies virus, Trypanosoma cruzi and / or Chergos disease, Ebola Viruses, Plasmodium falciparum, Marburg virus, Japanese encephalitis virus, St. Louis encephalitis virus, West Nile virus, yellow fever virus, Bacillus anthracis, botulinum toxin, ricin or Shiga toxin and / or Shiga-like toxin.

[0044] In one aspect, exposure is enteral (to the intestines), gastrointestinal tract, epidural (to the dura mater), oral (by means of the mouth), transdermal, intracerebral (to the brain), ventricle Intranasal (into the ventricles of the brain), epidermal (into the skin), intradermal (into the skin itself), subcutaneous (into the skin), nasal administration (through the nose), intravenous (into the veins), intravenous Intra-bolus injection, intravenous drip, intra-arterial (into the artery), intramuscular (into the muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal) ), intraparenchymal (into the brain tissue), intraperitoneal (infusion or injection into the peritoneum), intravesical infusion, intravitreal (through the eye), intracavernous sinus injection (into the pathological cavity), intracavity (into the pathological cavity) in the base of the penis), intravaginal administration, intrauterine, extraamniotic administration, transdermal (diffusion through intact skin for systemic dispersion), transmucosal (diffusion through mucous membranes), transvaginal, insufflation (sniffing), sublingual, Sublabial, enema, eye drops (to the conjunctiva), ear drops, transaural (in or by the ear), transbuccal (directed toward the cheek), transconjunctival, skin, teeth (to one or Multiple teeth), electroosmosis, intracervical, intrasinusal, intratracheal, extracorporeal, hemodialysis, infiltrative, interstitial, intraabdominal, intraamniotic, intraarticular, intrabiliary, intrabronchial, intracystic, intrachondral ( In cartilage), in the tail (in the tail of equids), in the cistern (in the cisterna magna), in the cornea (in the cornea), in the dental crown, in the coronary artery (in the coronary artery), in the penile sponge In vivo (in the expandable space of the corpus cavernosum of the penis), intradiscal (in the intervertebral disc), intraductal (in the glandular duct), intraduodenal (in the duodenum), intradural (in the dura mater) or below it), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (in the stomach), intragingival (in the gums), intraileal (in the distal part of the small intestine), intralesional ( Within the localized lesion or directly introduced to the localized lesion), intracavitary (within the lumen of the tube), intralymphatic (within the lymph), intramedullary (within the marrow cavity of the bone), intrameningeal (within the meninges) , intramyocardial (within the heart muscle), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura), intraprostatic (within the prostate gland) ), intrapulmonary (in the lungs or their bronchi), intrasinus (in the paranasal sinus or periorbital sinus), intraspinal (in the spinal column), intrasynovial (in the synovial fluid cavity of the joint), intratendinous (in the synovial fluid cavity of the joint) Intratendinous), intratesticular (inside the testis), intrathecal (in the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (inside the chest), intracanalicular (in the small tubes of the organ), intratumoral (within a tumor), intratympanic (within the tympanic cavity (aurus media)), intravascular (within one or more blood vessels), intraventricular (within a room), iontophoresis (in which ions of soluble salts move by means of an electric current) into body tissues), lavage (flushing or flushing of an open wound or body cavity), translaryngeal (directly to the throat), nasogastric tube (through the nose and into the stomach), occlusive dressing technique (administered locally, followed by other Covered by a dressing covering an occlusive area), transocular (to the outside of the eye), transoropharyngeal (directly to the mouth and pharynx), parenteral, transcutaneous, periarticular, epidural, perineural, periodontal, transrectal, Respiratory tract (by oral or nasal inhalation into the respiratory tract for local or systemic effects), retrobulbar (retropontine or retrobulbar), soft tissue, subarachnoid, subconjunctival, submucosal, surface, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (across or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), transvaginal, caudal block (caudal block) , diagnosis, nerve block, biliary perfusion, cardiac perfusion, photoremoval or transspinal. Simple explanation of the diagram

[0045] [picture] [1] is a diagram illustrating an embodiment of the tropism exploration platform of the present invention.

[0046] [picture] [2] is a diagram illustrating the initial polynucleotide construct of the present invention, which can be linear or cyclic.

[0047] [picture] [3A] is a diagram illustrating a series of reference polynucleotide constructs of the present invention, which may include at least a barcode region (BC) and / or a reverse barcode region (CB) and a payload region (P ).

[0048] [picture] [3B] is a diagram illustrating a series of reference polynucleotide constructs of the present invention, in which the barcode region (BC) or the reverse barcode region (CB) can overlap the payload region (P).

[0049] [picture] [3C] is a diagram illustrating a series of reference polynucleotide constructs of the present invention, which may include at least one tag and / or label.

[0050] [picture] [4A] is a diagram illustrating a series of cyclic reference polynucleotide constructs of the present invention, which may include at least one barcode region (BC) and / or a reverse barcode region (CB) and a payload region (P) .

[0051] [picture] [4B] is a diagram illustrating a series of cyclic reference polynucleotide constructs of the present invention, in which the barcode region (BC) or the reverse barcode region (CB) can overlap the payload region (P).

[0052] [picture] [4C] is a diagram illustrating a series of reference polynucleotide constructs of the present invention, which may include at least one tag and / or label.

[0053] [picture] [5] is a diagram illustrating a series of delivery vehicles of the present invention. Implementation

[0054] Cross-references to related applications

[0055] This application claims the rights and priority of U.S. Provisional Patent Application No. 63 / 293,289 filed on December 23, 2021, the contents of which are incorporated herein by reference in full. Sequence listing reference

[0056] This application contains a sequence listing, which has been submitted electronically in XML format. The sequence listing XML is incorporated by reference in its entirety. The XML file created on December 22, 2022 is named REG-007WO.XML and has a size of 98,807 bytes. I. Introduction to Tropical Delivery Systems

[0057] Considering its versatility, lower immune response and higher efficacy compared with traditional therapies, nucleic acid therapy has become the main method for treating various diseases and treatment indications. For example, nucleic acid therapeutics include the use of: small interfering RNA (siRNA), to reduce the translation of messenger RNA (mRNA); mRNA, as a means to produce the target of interest; circular RNA (oRNA), which can provide Continuous production of polypeptides or peptides may be sponges that compete with other RNA molecules; and viral vectors are used to provide continuous production of a target of interest. However, some nucleic acids are unstable and prone to degradation, so they need to be formulated to prevent degradation and aid intracellular delivery of the nucleic acid.

[0058] Current delivery vehicles, including lipid-based delivery vehicles such as lipid nanoparticles and liposomes, focus on protecting the carrier, but do not focus on positioning the delivery vehicle or delivering the vehicle to specific areas in the body.

[0059] This article provides a tropism discovery platform for the evaluation of targeting systems for localized delivery to specific target regions, cells, or tissues. like [picture] As shown in [1], the tropism discovery platform can be used to evaluate lipid nanoparticle (LNP) libraries and / or AAV libraries to determine the tropism or signature profile of the targeting systems in the library. The depot can be administered to an individual (e.g., a non-human primate, rabbit, mouse, rat, or another mammal), and the individual's organs and tissues scanned and / or collected and analyzed to determine the LNPs in the depot or the location of identifiers (such as barcodes, tags, signals and / or labels) contained in or associated with the AAV. This analysis provides the tropism signature or profile of each LNP and AAV in the library. Initial structure architecture

[0060] The targeting system of the tropism discovery platform may include coding or an initial construct including a cargo or payload. Initial polynucleotide construct that can be linear or circular

[0100] Examples are provided in [picture] [2] in. Initial polynucleotide construct May include at least one payload area for or encoding the payload or bearer of interest

[10] . Initial polynucleotide construct

[0100] May contain 1 or 2 flanking areas

[20] , and flanking area 2 [0] can be located in the payload area

[10] 5' or payload area

[10] -3'. In some cases, the initial polynucleotide construct

[0100] Does not contain side area 2 [0]. Initial polynucleotide construct

[0100] side area

[20] May include at least one adjustment area

[30] . Initial polynucleotide construct

[0100] at least one side area

[20] May include at least one identifier area

[40] . identifier area

[40] Can be, but is not limited to, barcodes, tags, signals and / or labels. In addition, the identifier area

[40] Can be located in the payload area

[10] may be located within the payload area

[10] and at least one flanking area

[20] .

[0061] In some embodiments, the initial construct includes about 5 to about 10,000 residues. As a non-limiting example, the length of the initial construct may be 5 to 30, 5 to 50, 5 to 100, 5 to 250, 5 to 500, 5 to 1,000, 5 to 1,500, 5 to 3,000, 5 to 5,000, 5 to 7,000, 5 to 10,000, 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 1,000, 30 to 1,500, 30 to 3,000, 30 to 5,000, 30 to 7,000, 30 to 10,000, 100 to 250, 100 to 500, 100 to 1,000, 100 to 1,500, 100 to 3,000, 100 to 5,000, 100 to 7,000, 100 to 10,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 500 to 5,00 0. 500 to 7,000, 500 to 10,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 5,000, 1,000 to 7,000, 1,000 to 10,000, 1,500 to 3,000, 1,500 to 5,000, 1,5 00 to 7,000, 1,500 to 10,000, 2,000 to 3,000, 2,000 to 5,000, 2,000 to 7,000, 2,000 to 10,000, 3,000 to 5,000, 3,000 to 7,000, 3,000 to 10,000, 5,000 to 7,000, 5,000 to 10,000 and 7,000 to 10,000.

[0062] In some embodiments, the payload region is greater than about 5 residues in length, such as, but not limited to, at least or greater than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000 or greater than 10,000 residues.

[0063] In some embodiments, the length of the flanking regions may independently range from 0 to 10,000 residues, such as, but not limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,40 ,45,50,55,60,70,80,90,100,120,140,160,180,200,250,300,350,400,450,500,600,700,800,900,1,000,1,100 , 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000 and 10,000.

[0064] In some embodiments, the length of the regulatory region may independently range from 0 to 3,000 residues, such as, but not limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500 and 3,000.

[0065] In some embodiments, the initial construct can be cyclized or concatenated to create molecules that assist in the interaction between the 3' and 5' ends of the initial construct. baseline architecture

[0066] The initial architecture that includes at least one identifier (eg, barcode, tag, signal, and / or label) is called the base architecture. The reference polynucleotide construct may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more identifiers that may be present throughout the reference polynucleotide construct. Same or different.

[0067] In some embodiments, the length of the identifier region may independently range from 1 to 3,000 residues, such as, but not limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,40 ,45,50,55,60,70,80,90,100,120,140,160,180,200,250,300,350,400,450,500,600,700,800,900,1,000,1,100 , 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500 and 3,000. As non-limiting examples, the identifier region may be 1-5 residues, 2-5 residues, 3-5 residues, 2-7 residues, 3-7 residues, 1-5 residues in length. 10 residues, 2-10 residues, 3-10 residues, 5-10 residues, 7-10 residues, 1-15 residues, 2-15 residues, 3-15 residues, 5-15 residues, 7-15 residues, 10-15 residues, 12-15 residues, 1-20 residues, 2-20 residues, 3-20 Residues, 5-20 residues, 7-20 residues, 10-20 residues, 12-20 residues, 15-20 residues, 17-20 residues, 1-25 residues base, 2-25 residues, 3-25 residues, 5-25 residues, 7-25 residues, 10-25 residues, 12-25 residues, 15-25 residues , 17-25 residues, 20-25 residues, 1-30 residues, 2-30 residues, 3-30 residues, 5-30 residues, 7-30 residues, 10-30 residues, 12-30 residues, 15-30 residues, 17-30 residues, 20-30 residues, 25-30 residues, 1-35 residues, 2 -35 residues, 3-35 residues, 5-35 residues, 7-35 residues, 10-35 residues, 12-35 residues, 15-35 residues, 17- 35 residues, 20-35 residues, 25-35 residues, 30-35 residues, 1-35 residues, 2-35 residues, 3-35 residues, 5-35 residues, 7-35 residues, 10-35 residues, 12-35 residues, 15-35 residues, 17-35 residues, 20-35 residues, 25-35 Residues, 30-35 residues, 1-40 residues, 2-40 residues, 3-40 residues, 5-40 residues, 7-40 residues, 10-40 residues base, 12-40 residues, 15-40 residues, 17-40 residues, 20-40 residues, 25-40 residues, 30-40 residues, 35-40 residues , 1-45 residues, 2-45 residues, 3-45 residues, 5-45 residues, 7-45 residues, 10-45 residues, 12-45 residues, 15-45 residues, 17-45 residues, 20-45 residues, 25-45 residues, 30-45 residues, 35-45 residues, 40-45 residues, 1 -50 residues, 2-50 residues, 3-50 residues, 5-50 residues, 7-50 residues, 10-50 residues, 12-50 residues, 15- 50 residues, 17-50 residues, 20-50 residues, 25-50 residues, 30-50 residues, 35-50 residues, 40-50 residues or 45-50 residues.

[0068] Non-limiting examples of reference polynucleotide constructs having at least one identifier, which may be linear or cyclic, are provided in [picture] [3A] [,picture] [3B] and [picture] [3C]. Non-limiting examples of cyclic reference polynucleotide constructs having at least one identifier are provided in [picture] [4A] [,picture] [4B] and [picture] [4C]. exist [picture] [3A] [,picture] [3B] [,picture] [4A] and [picture] In [4B], the reference polynucleotide construct includes a payload region (referred to as "P" in the figure) and at least one identifier region (referred to as "BC" in the figure) and / or a reverse identifier region (Referred to as "CB" in the diagram). exist [picture] [3C] and [picture] In [4C], the reference polynucleotide construct includes a payload region (referred to as "P" in the figure) and at least one identifier portion associated with the reference polynucleotide construct.

[0069] In some embodiments, the identifier area in the base construct overlaps the payload area. As used herein, "overlap" means that at least one nucleotide of the identifier region extends into the payload region. In some aspects, the identifier region overlaps the payload region by 1, 2, 3, 4, 5, 6, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides Nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleosides acid, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides Nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 ​​nucleosides acid, 49 nucleotides, 50 nucleotides or more than 50 nucleotides. In some aspects, the identifier region overlaps the payload region by 1-5 nucleotides, 2-5 nucleotides, 3-5 nucleotides, 2-7 nucleotides, 3-7 nucleotides, Nucleotides, 1-10 nucleotides, 2-10 nucleotides, 3-10 nucleotides, 5-10 nucleotides, 7-10 nucleotides, 1-15 nucleosides Acid, 2-15 nucleotides, 3-15 nucleotides, 5-15 nucleotides, 7-15 nucleotides, 10-15 nucleotides, 12-15 nucleotides, 1-20 nucleotides, 2-20 nucleotides, 3-20 nucleotides, 5-20 nucleotides, 7-20 nucleotides, 10-20 nucleotides, 12- 20 nucleotides, 15-20 nucleotides, 17-20 nucleotides, 1-25 nucleotides, 2-25 nucleotides, 3-25 nucleotides, 5-25 nucleotides Nucleotides, 7-25 nucleotides, 10-25 nucleotides, 12-25 nucleotides, 15-25 nucleotides, 17-25 nucleotides, 20-25 nucleosides Acid, 1-30 nucleotides, 2-30 nucleotides, 3-30 nucleotides, 5-30 nucleotides, 7-30 nucleotides, 10-30 nucleotides, 12-30 nucleotides, 15-30 nucleotides, 17-30 nucleotides, 20-30 nucleotides, 25-30 nucleotides, 1-35 nucleotides, 2- 35 nucleotides, 3-35 nucleotides, 5-35 nucleotides, 7-35 nucleotides, 10-35 nucleotides, 12-35 nucleotides, 15-35 Nucleotides, 17-35 nucleotides, 20-35 nucleotides, 25-35 nucleotides, 30-35 nucleotides, 1-35 nucleotides, 2-35 nucleosides Acid, 3-35 nucleotides, 5-35 nucleotides, 7-35 nucleotides, 10-35 nucleotides, 12-35 nucleotides, 15-35 nucleotides, 17-35 nucleotides, 20-35 nucleotides, 25-35 nucleotides, 30-35 nucleotides, 1-40 nucleotides, 2-40 nucleotides, 3- 40 nucleotides, 5-40 nucleotides, 7-40 nucleotides, 10-40 nucleotides, 12-40 nucleotides, 15-40 nucleotides, 17-40 Nucleotides, 20-40 nucleotides, 25-40 nucleotides, 30-40 nucleotides, 35-40 nucleotides, 1-45 nucleotides, 2-45 nucleosides acid, 3-45 nucleotides, 5-45 nucleotides, 7-45 nucleotides, 10-45 nucleotides, 12-45 nucleotides, 15-45 nucleotides, 17-45 nucleotides, 20-45 nucleotides, 25-45 nucleotides, 30-45 nucleotides, 35-45 nucleotides, 40-45 nucleotides, 1- 50 nucleotides, 2-50 nucleotides, 3-50 nucleotides, 5-50 nucleotides, 7-50 nucleotides, 10-50 nucleotides, 12-50 nucleotides Nucleotides, 15-50 nucleotides, 17-50 nucleotides, 20-50 nucleotides, 25-50 nucleotides, 30-50 nucleotides, 35-50 nucleosides acid, 40-50 nucleotides or 45-50 nucleotides.

[0070] In some embodiments, the reference polynucleotide construct includes a payload region and an identifier region. The identifier region may be located 5' from the payload region, 3' from the payload region, or the identifier region may overlap the 5' end or the 3' end of the payload region.

[0071] In some embodiments, the reference polynucleotide construct includes a payload region and two identifier regions. Each identifier region may be independently located 5' from the payload region, 3' from the payload region, or the identifier region may overlap with the 5' end or the 3' end of the payload region.

[0072] As a non-limiting example, the first identifier area is located 5' from the payload area and the second identifier area is located 3' from the payload area. As a non-limiting example, the first and second identifier areas are located 5' of the payload area. As a non-limiting example, the first and second identifier areas are located 3' of the payload area.

[0073] As a non-limiting example, the first identifier area is reversed and located 5' of the payload area, and the second identifier area is located 3' of the payload area. As a non-limiting example, the first identifier area is inverted and located 5' of the payload area, and the second identifier area is inverted and located 3' of the payload area. As a non-limiting example, the first identifier area is located 5' of the payload area, and the second identifier area is reversed and located 3' of the payload area. As a non-limiting example, the first and second identifier areas are both inverted and located 5' of the payload area. As a non-limiting example, the first and second identifier areas are located 5' of the payload area, and the first identifier area is inverted. As a non-limiting example, the first and second identifier areas are located 5' of the payload area, and the second identifier area is inverted. As a non-limiting example, the first and second identifier areas are both inverted and located 3' of the payload area. As a non-limiting example, the first and second identifier areas are located 3' of the payload area, and the first identifier area is inverted. As a non-limiting example, the first and second identifier areas are located 3' of the payload area, and the second identifier area is inverted.

[0074] As a non-limiting example, the first identifier area is located 5' from the payload area and overlaps the payload area, and the second identifier area is located 3' from the payload area. As a non-limiting example, the first identifier area is located 5' from the payload area, and the second identifier area is located 3' from the payload area and overlaps the payload area.

[0075] As a non-limiting example, the first and second identifier areas are located 5' of the payload area, and the second identifier area overlaps the payload area. As a non-limiting example, the first and second identifier areas are located 3' of the payload area, and the first identifier area overlaps the payload area.

[0076] As a non-limiting example, the first identifier area is inverted, located 5' from and overlapping the payload area, and the second identifier area is located 3' from the payload area. As a non-limiting example, the first identifier area is inverted and located 5' from the payload area, and the second identifier area is located 3' from the payload area and overlaps the payload area. As a non-limiting example, the first identifier area is inverted and located 5' from the payload area, the second identifier area is located 3' from the payload area, and both the first and second identifier areas are identical to Payload areas overlap.

[0077] As a non-limiting example, the first identifier area is inverted, located 5' from the payload area and overlaps the payload area, and the second identifier area is inverted and located 3' from the payload area. As a non-limiting example, the first identifier area is inverted and located 5' from the payload area, and the second identifier area is inverted, located 3' from the payload area and overlaps the payload area. As a non-limiting example, the first identifier area is inverted and located 5' of the payload area, and the second identifier area is inverted and located 3' of the payload area, and the first and second identification The descriptor area both overlaps with the payload area.

[0078] As a non-limiting example, the first identifier area is located 5' from the payload area and overlaps the payload area, and the second identifier area is inverted and located 3' from the payload area. As a non-limiting example, the first identifier area is located 5' from the payload area, and the second identifier area is inverted, located 3' from the payload area and overlaps the payload area. As a non-limiting example, the first identifier area is located 5' from the payload area, and the second identifier area is inverted and located 3' from the payload area, and both the first and second identifier areas are Overlaps the payload area.

[0079] As a non-limiting example, the first and second identifier areas are both inverted and located 5' of the payload area, and the second identifier area overlaps the payload area. As a non-limiting example, the first and second identifier areas are located 5' from the payload area, the first identifier area is reversed, and the second identifier area overlaps the payload area. As a non-limiting example, the first and second identifier areas are located 5' of the payload area, and the second identifier area is inverted and overlaps the payload area. As a non-limiting example, both the first and second identifier areas are inverted and located 3' of the payload area, and the first identifier area overlaps the payload area. As a non-limiting example, the first and second identifier areas are located in the payload area 3', and the first identifier area is inverted and overlaps the payload area. As a non-limiting example, the first and second identifier areas are located 3' of the payload area, the second identifier area is reversed, and the first payload area overlaps the payload area.

[0080] In some embodiments, at least one identifier moiety can be combined with a reference polynucleotide construct. The reference polynucleotide construct may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more identifier moieties associated with the reference polynucleotide construct, which may be The same moiety or a different moiety that binds to the reference polynucleotide construct. Each identifier portion may be independently located 5' flanking the payload area, 3' flanking the payload area, or the identifier portion may be positioned across the payload area and 5' flanking the area. end or 3' end. In some aspects, the location of the identifier portion may include one or more nucleotides in the payload region, such as, but not limited to, 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, nucleotide, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides , 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 Nucleotides, 22 Nucleotides, 23 Nucleotides, 24 Nucleotides, 25 Nucleotides, 26 Nucleotides, 27 Nucleotides, 28 Nucleotides, 29 Nucleotides nucleotide, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides , 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 ​​nucleotides, 49 nucleotides, 50 nucleotides or more than 50 nucleotides. In some aspects, the location of the identifier portion may include one or more nucleotides in the payload region, such as, but not limited to, 1-5 nucleotides, 2-5 nucleotides, 3-5 nucleotides, nucleotides, 2-7 nucleotides, 3-7 nucleotides, 1-10 nucleotides, 2-10 nucleotides, 3-10 nucleotides, 5-10 nucleotides , 7-10 nucleotides, 1-15 nucleotides, 2-15 nucleotides, 3-15 nucleotides, 5-15 nucleotides, 7-15 nucleotides, 10 -15 nucleotides, 12-15 nucleotides, 1-20 nucleotides, 2-20 nucleotides, 3-20 nucleotides, 5-20 nucleotides, 7-20 nucleotides, 10-20 nucleotides, 12-20 nucleotides, 15-20 nucleotides, 17-20 nucleotides, 1-25 nucleotides, 2-25 nuclei nucleotides, 3-25 nucleotides, 5-25 nucleotides, 7-25 nucleotides, 10-25 nucleotides, 12-25 nucleotides, 15-25 nucleotides , 17-25 nucleotides, 20-25 nucleotides, 1-30 nucleotides, 2-30 nucleotides, 3-30 nucleotides, 5-30 nucleotides, 7 -30 nucleotides, 10-30 nucleotides, 12-30 nucleotides, 15-30 nucleotides, 17-30 nucleotides, 20-30 nucleotides, 25-30 nucleotides, 1-35 nucleotides, 2-35 nucleotides, 3-35 nucleotides, 5-35 nucleotides, 7-35 nucleotides, 10-35 nuclei nucleotides, 12-35 nucleotides, 15-35 nucleotides, 17-35 nucleotides, 20-35 nucleotides, 25-35 nucleotides, 30-35 nucleotides , 1-35 nucleotides, 2-35 nucleotides, 3-35 nucleotides, 5-35 nucleotides, 7-35 nucleotides, 10-35 nucleotides, 12 -35 nucleotides, 15-35 nucleotides, 17-35 nucleotides, 20-35 nucleotides, 25-35 nucleotides, 30-35 nucleotides, 1-40 nucleotides, 2-40 nucleotides, 3-40 nucleotides, 5-40 nucleotides, 7-40 nucleotides, 10-40 nucleotides, 12-40 nuclei nucleotides, 15-40 nucleotides, 17-40 nucleotides, 20-40 nucleotides, 25-40 nucleotides, 30-40 nucleotides, 35-40 nucleotides , 1-45 nucleotides, 2-45 nucleotides, 3-45 nucleotides, 5-45 nucleotides, 7-45 nucleotides, 10-45 nucleotides, 12 -45 nucleotides, 15-45 nucleotides, 17-45 nucleotides, 20-45 nucleotides, 25-45 nucleotides, 30-45 nucleotides, 35-45 nucleotides, 40-45 nucleotides, 1-50 nucleotides, 2-50 nucleotides, 3-50 nucleotides, 5-50 nucleotides, 7-50 nuclei nucleotides, 10-50 nucleotides, 12-50 nucleotides, 15-50 nucleotides, 17-50 nucleotides, 20-50 nucleotides, 25-50 nucleotides , 30-50 nucleotides, 35-50 nucleotides, 40-50 nucleotides or 45-50 nucleotides.

[0081] In some embodiments, an identifier moiety can be combined with a reference polynucleotide construct. As a non-limiting example, the identifier moiety may bind to the reference polynucleotide construct on the 5' end of the reference polynucleotide construct. As a non-limiting example, the identifier moiety can bind to the reference polynucleotide construct on the 5' flanking region. As a non-limiting example, the identifier moiety can bind to the reference polynucleotide construct on the 3' flanking region. As a non-limiting example, the identifier moiety can bind to the reference polynucleotide construct on the 3' end of the reference polynucleotide construct. As a non-limiting example, the identifier portion can be combined with a reference polynucleotide construct on the payload region. As a non-limiting example, the reference polynucleotide construct includes an identifier portion positioned across the 5' end and 5' flanking regions of the payload region. As a non-limiting example, the reference polynucleotide construct includes an identifier portion positioned across the 3' end and 3' flanking regions of the payload region.

[0082] In some embodiments, two identifier moieties are bound to a reference polynucleotide construct. As a non-limiting example, the first identifier portion and the second identifier portion are located on the 5' flanking region. As a non-limiting example, the first identifier portion and the second identifier portion are located on the payload area. As a non-limiting example, the first identifier portion and the second identifier portion are located on the 3' flanking region. As a non-limiting example, the first identifier portion and the second identifier portion are located on the 5' end of the reference polynucleotide construct. As a non-limiting example, the first identifier portion and the second identifier portion are located on the 3' end of the reference polynucleotide construct.

[0083] As a non-limiting example, a first identifier portion is located on the 5' end of the reference polynucleotide construct and a second identifier portion is located on the 5' flanking region. As a non-limiting example, the first identifier portion is located on the 5' end of the reference polynucleotide construct and the second identifier portion is located on the payload region. As a non-limiting example, a first identifier portion is located on the 5' end of the reference polynucleotide construct and a second identifier portion is located on the 3' flanking region. As a non-limiting example, the first identifier portion is located on the 5' end of the reference polynucleotide construct, and the second identifier portion is positioned across the 5' flanking region and the payload region. As a non-limiting example, the first identifier portion is located on the 5' end of the reference polynucleotide construct, and the second identifier portion is positioned across the 3' flanking region and the payload region. As a non-limiting example, the first identifier portion is located on the 5' end of the reference polynucleotide construct and the second identifier portion is located on the 3' end of the reference polynucleotide construct.

[0084] As a non-limiting example, the first identifier portion is located on the 5' flanking region and the second identifier portion is located on the payload region. As a non-limiting example, the first identifier portion is located on the 5' flanking region and the second identifier portion is located on the 3' flanking region. As a non-limiting example, the first identifier portion is located on the 5' flanking region, and the second identifier portion is positioned across the 5' flanking region and the payload region. As a non-limiting example, the first identifier portion is located on the 5' flanking region, and the second identifier portion is positioned across the 3' flanking region and the payload region. As a non-limiting example, a first identifier portion is located on the 5' flanking region and a second identifier portion is located on the 5' end of the reference polynucleotide construct. As a non-limiting example, a first identifier portion is located on the 5' flanking region and a second identifier portion is located on the 3' end of the reference polynucleotide construct.

[0085] As a non-limiting example, the first identifier portion is positioned across the 5' flanking region and the payload region, and the second identifier portion is located on the 5' end of the reference polynucleotide construct. As a non-limiting example, the first identifier portion is positioned across the 5' flanking region and the payload region, and the second identifier portion is located on the 5' flanking region. As a non-limiting example, the first identifier portion is positioned across the 5' flanking region and the payload region, and the second identifier portion is located on the payload region. As a non-limiting example, the first identifier portion is positioned across the 5' flanking region and the payload region, and the second identifier portion is positioned across the 3' flanking region and the payload region. As a non-limiting example, the first identifier portion is positioned across the 5' flanking region and the payload region, and the second identifier portion is located on the 3' flanking region. As a non-limiting example, the first identifier portion is positioned across the 5' flanking region and the payload region, and the second identifier portion is located on the 3' end of the reference polynucleotide construct.

[0086] As a non-limiting example, the first identifier portion is located on the payload region and the second identifier portion is located on the 5' end of the reference polynucleotide construct. As a non-limiting example, the first identifier portion is located on the payload area and the second identifier portion is located on the 5' flanking area. As a non-limiting example, the first identifier portion is located on the payload area, and the second identifier portion is positioned across the 5' flanking area and the payload area. As a non-limiting example, the first identifier portion is located on the payload area, and the second identifier portion is positioned across the 3' flanking area and the payload area. As a non-limiting example, the first identifier portion is located on the payload area and the second identifier portion is located on the 3' flanking area. As a non-limiting example, the first identifier portion is located on the payload region and the second identifier portion is located on the 3' end of the reference polynucleotide construct.

[0087] As a non-limiting example, the first identifier portion is positioned across the 3' flanking region and the payload region, and the second identifier portion is located on the 5' end of the reference polynucleotide construct. As a non-limiting example, the first identifier portion is positioned across the 3' flanking region and the payload region, and the second identifier portion is located on the 5' flanking region. As a non-limiting example, the first identifier portion is positioned across the 3' flanking region and the payload region, and the second identifier portion is positioned across the 5' flanking region and the payload region. As a non-limiting example, the first identifier portion is positioned across the 3' flanking region and the payload region, and the second identifier portion is located on the payload region. As a non-limiting example, the first identifier portion is positioned across the 3' flanking region and the payload region, and the second identifier portion is located on the 3' flanking region. As a non-limiting example, the first identifier portion is positioned across the 3' flanking region and the payload region, and the second identifier portion is located on the 3' end of the reference polynucleotide construct.

[0088] As a non-limiting example, the first identifier portion is positioned across the 3' flanking region and the payload region, and the second identifier portion is located on the 5' flanking region. As a non-limiting example, the first identifier portion is positioned across the 5' flanking region and the payload region, and the second identifier portion is located on the payload region. As a non-limiting example, the first identifier portion is positioned across the 5' flanking region and the payload region, and the second identifier portion is positioned across the 3' flanking region and the payload region. As a non-limiting example, the first identifier portion is positioned across the 5' flanking region and the payload region, and the second identifier portion is located on the 3' flanking region. As a non-limiting example, the first identifier portion is positioned across the 5' flanking region and the payload region, and the second identifier portion is located on the 3' end of the reference polynucleotide construct.

[0089] As a non-limiting example, a first identifier portion is located on the 3' flanking region and a second identifier portion is located on the 5' end of the reference polynucleotide construct. As a non-limiting example, the first identifier portion is located on the 3' flanking region and the second identifier portion is located on the 5' flanking region. As a non-limiting example, the first identifier portion is located on the 3' flanking region, and the second identifier portion is positioned across the 5' flanking region and the payload region. As a non-limiting example, the first identifier portion is located on the 3' flanking region and the second identifier portion is located on the payload region. As a non-limiting example, the first identifier portion is located on the 3' flanking region, and the second identifier portion is positioned across the 3' flanking region and the payload region. As a non-limiting example, a first identifier portion is located on the 3' flanking region and a second identifier portion is located on the 3' end of the reference polynucleotide construct.

[0090] As a non-limiting example, the first identifier portion is located on the 3' end of the reference polynucleotide construct and the second identifier portion is located on the 5' end of the reference polynucleotide construct. As a non-limiting example, a first identifier portion is located on the 3' end of the reference polynucleotide construct and a second identifier portion is located on the 5' flanking region. As a non-limiting example, the first identifier portion is located on the 5' end of the reference polynucleotide construct, and the second identifier portion is positioned across the 5' flanking region and the payload region. As a non-limiting example, the first identifier portion is located on the 3' end of the reference polynucleotide construct and the second identifier portion is located on the payload region. As a non-limiting example, the first identifier portion is located on the 5' end of the reference polynucleotide construct, and the second identifier portion is positioned across the 3' flanking region and the payload region. As a non-limiting example, a first identifier portion is located on the 3' end of the reference polynucleotide construct and a second identifier portion is located on the 3' flanking region.

[0091] In some embodiments, three identifier moieties are combined with a reference polynucleotide construct.

[0092] In some embodiments, four identifier moieties are combined with a reference polynucleotide construct.

[0093] In some embodiments, five identifier moieties are combined with a reference polynucleotide construct.

[0094] In some embodiments, six identifier moieties are combined with a reference polynucleotide construct.

[0095] In some embodiments, seven identifier moieties are combined with a reference polynucleotide construct.

[0096] In some embodiments, eight identifier moieties are combined with a reference polynucleotide construct.

[0097] In some embodiments, nine identifier moieties are combined with a reference polynucleotide construct.

[0098] In some embodiments, ten identifier moieties are combined with a reference polynucleotide construct. II. Carrier and payload

[0099] The initial and baseline constructs of the present invention may contain, encode or be combined with carriers or payloads. As used herein, the term "cargo" or "payload" may refer to one or more molecules or structures included in a delivery vehicle used to deliver a cell or tissue to a cell or tissue. Non-limiting examples of carriers may include nucleic acids, polypeptides, peptides, proteins, liposomes, labels, tags, small chemical molecules, large biological molecules, and any combination or fragment thereof. In the initial structure and the base structure, the area of ​​the structure that contains or encodes the load or payload is called the "cargo area" or "payload area."

[0100] In some embodiments, the carrier or payload is or encodes a biologically active molecule, such as, but not limited to, a therapeutic protein. As used herein, the term "biological activity" refers to any property of an agent that is active in a biological system, and specifically in an organism. For example, an agent is considered to be biologically active if it has a biological effect on an organism when administered to that organism. In some embodiments, the carrier or payload is or encodes one or more prophylactically or therapeutically active proteins, polypeptides, or other factors. As a non-limiting example, the carrier or payload may be or encode an agent that enhances tumor killing activity in cancer, such as, but not limited to, TRAIL or tumor necrosis factor (TNF). As another non-limiting example, the carrier or payload may be or encode an agent suitable for treating a condition such as muscular dystrophy (e.g., the carrier or payload is or encodes myosin), cardiovascular disease (e.g., the carrier or payload is or encodes myosin), cardiovascular disease (e.g., the carrier The carrier or payload is or encodes SERCA2a, GATA4, Tbx5, Mef2C, Hand2, Myocd, etc.), neurodegenerative diseases (for example, the carrier or payload is or encodes NGF, BDNF, GDNF, NT-3, etc.), chronic pain ( For example, the carrier or payload is or encodes GlyRal), enkephalin or glutamate decarboxylase (eg, the carrier or payload is or encodes GAD65, GAD67 or another isoform), lung disease (eg the carrier or payload is or encodes GAD65, GAD67 or another isoform), lung disease (eg the carrier or payload is or encodes GlyRal) is or encodes CFTR), hemophilia (e.g., the carrier or payload is or encodes factor VIII or factor IX), neoplasia (e.g., the carrier or payload is or encodes PTEN, ATM, ATR, EGFR, ERBB2, ERBB3, ERBB4, Notchl, Notch2, Notch3, Notch4, AKT, AKT2, AKT3, HIF, HI Fla, HIF3a, Met, HRG, Bcl2, PPARα, PPARγ, WT1 (Wilms Tumor), FGF receptor Family members (5 members: 1, 2, 3, 4, 5), CDKN2a, APC, RB (retinoblastoma), MEN1, VHL, BRCA1, BRCA2, AR (androgen receptor), TSG101, IGF, IGF receptor, Igfl (4 variants), Igf2 (3 variants), Igfl receptor, IGF2 receptor, Bax, Bcl2, apoptotic protease family (9 members: 1, 2, 3, 4, 6 , 7, 8, 9, 12), Kras, Ape), age-related macular degeneration (for example, the carrier or payload is or encodes Aber, Ccl2, Cc2, cp (ceruloplasmin), Timp3, cathepsin D, Vldlr), schizophrenia (such as neuregulin (Nrgl), Erb4 (neuregulin receptor), complexin-l (Cplxl), Tphl tryptophan hydroxylase, Tph2 tryptophan hydroxylase 2, Axonin 1, GSK3, GSK3a, GSK3b, 5-HIT (Slc6a4), COMT, DRD (Drdla), SLC6A3, DAOA, DTNBPI, Dao (Daol)), trinucleotide repeat disorders such as HTT (Huntington's ( Huntington's) Dx), SBMA / SMAXI / AR (Kennedy's) Dx), FXN / X25 (Friedrich's Ataxia), ATX3 (Machado-Joseph's) Dx), ATXNI and ATXN2 (spinocerebellar ataxia), DMPK (myotonic dystrophy), atrophin-1 and Atnl (DRPLA Dx), CBP (Creb-BP-global instability), VLDLR (AZ Alzheimer's), Atxn7, Atxn10), Fragile APH-1 (alpha and beta), presenilin (Psenl), ncstn (Ncstn), PEN-2), ALS (e.g., the carrier or payload is or encodes SOD1, ALS2, STEX, FUS, TARD BP, VEGF ( VEGF-a, VEGF-b, VEGF-c)), autism (e.g. the carrier or payload is or encodes Mecp2, BZRAP1, MDGA2, Sema5A, Axonin 1), Alzheimer's disease (e.g. the carrier The object or payload is or encodes El, CHIP, UCH, UBB, Tau, LRP, PICALM, Clusterin, PS1, SORL1, CR1, Vldlr, Ubal, Uba3, CHIP28 (Aqpl,Aquaporin 1), Uchll, Uchl3, APP), inflammation (e.g. the carrier or payload is or encodes IL-10, IL-1 (IL-Ia, IL-Ib), IL-13, IL-17 (IL -17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-171), 11-23, Cx3crl, ptpn22, TNFa, IBD’s NOD2 / CARD15, IL-6, IL-12 (IL-12a , IL-12b), CTLA4, Cx3cll), Parkinson's disease (e.g., x-synuclein, DJ-1, LRRK2, Parkin, PINK1), blood and coagulation disorders such as anemia, naked lymphocyte syndrome , bleeding disorders, hemophagocytic lymphohistiocytosis, hemophilia A, hemophilia B, bleeding disorders, leukopenia and disorders, sickle cell anemia and thalassemia (for example, the carrier or payload is Or codes CRAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH1, PSNI, RHAG, RH50A, NRAMP2, SPTB, ALAS2, ANH1, ASB, ABCB7, ABC7, ASAT, TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP, RFX5, TBXA2R, P2RX1, P2X1, HF1, CFH, HUS, MCFD2, FANCA, FAC A, FA1, FA, FA A, FAAP95, FAAP90, FLJ34064, FANCB, FANCC, FACC, BRCA2, FANCDI, FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, FHL3, F8, FSC, PI, ATT, F5, ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4, HBB, HBA2, HBB, HBD, LCRB, HBA1) , B-cell non-Hodgkin lymphoma or leukemia (e.g., the carrier or payload is or encodes BCL7A, BCL7, ALI, TCL5, SCL, TAL2, FLT3, NBS1, NBS, ZNFN1AI, 1KI, LYF1, HOXD4, HOX4B, BCR , CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AFIO, ARHGEF12, LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT, LPP, NPMI, NUP214, D9S46E, CAN , CAIN, RUNXI, CBFA2, AML1, WHSC1LI, NSD3, FLT3, AF1Q, NPMI, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF1Q, CALM, CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL , ALL, GRAF, NF1, VRNF, WSS, NFNS, PTPNII, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA, GATA1, GF1, ERYF1, NFE1, ABLI, NQO1, DIA4, NMOR1, NUP214, D9S46E , CAN, CAIN), inflammatory and immune-related diseases and disorders (e.g., the carrier or payload is or encodes KIR3DL1, NKAT3, NKB1, AMB11, K1R3DS1, IFNG, CXCL12, TNFRSF6, APT1, FAS, CD95, ALPS1A, IL2RG, SCIDX1 , SCIDX, IMD4, CCL5, SCYA5, D17S136E, TCP228, IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5), CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSFS, CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID, -13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-171), 11-23, Cx3crl, ptpn22, TNFa, NOD2 / CARD15 for IBD, IL -6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cII), JAK3, JAKL, DCLREIC, ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG , SCIDXI, SCIDX, IMD4), metabolic, liver, renal and protein diseases and disorders (e.g. the carrier or payload is or encodes TTR, PALB, APOA1, APP, AAA, CVAP, ADI, GSN, FGA, LYZ, TTR, PALB, KRT18, KRT8, CIRH1A, NAIC, TEX292, KIAA1988, CFTR, ABCC7, CF, MRP7, SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM, TCF1, HNF1A, MODY3, SCOD1, SCAl, CTNNB1, PDGFRL, PDGRL, PRLTS, AX1NI, AXIN, CTNNB1, TP53, P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5, UMOD, HNFJ, FJHN, MCKD2, ADMCKD2, PAH, PKU1, QDPR, DHPR, PTS, FCYT, PKHD1, ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63), muscle / skeletal diseases and disorders (e.g., the carrier or payload is or encodes DMD , BMD, MYF6, LMNA, LMN1, EMD2, FPLD, CMDIA, HGPS, LGMDIB, LMNA, LMNI, EMD2, FPLD, CMDIA, FSHMD1A, FSHD1A, FKRP, MDC1C, LGMD2I, LAMA2, LAMM, LARGE, KIAA0609, MDC1D, FCMD , TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B, SGCG, LGMD2C, DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E, SGCD, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRIM32 , HT2A, LGMD2H, FKRP, MDCIC, LGMD21, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1, LRP5, BMNDl, LRP7, LR3, OPPG, VBCH2, CLCN7 , CLC7, OPTA2, OSTMI, GL, TCIRG1, TIRC7, OC116, OPTB1, VAPB, VAPC, ALS8, SMN1, SMA1, SMA2, SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2, CATF1 , SMARD1), neurological and neuronal diseases and disorders (e.g., the carrier or payload is or encodes SOD1, ALS2, STEX, FUS, TARDBP, VEGF (VEGF-a, VEGF-b, VEGF-c), APP, AAA, CVAP, ADI, APOE, AD2, PSEN2, AD4, STM2, APBB2, FE65LI, NOS3, PLAU, URK, ACE, DCPI, ACEI, MPO, PAC1PI, PAXIPIL, PTIP, A2M, BLMH, BMH, PSEN1, AD3, Mecp2, BZRAP1, MDGA2, Sema5A, Neurexin 1, GLOl, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2, FMR2, FXR1, FXR2, mGLUR5, HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2 , TBP, SCA17, NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJI, PARK7, LRRK2, PARK8, PINK1, PARK6, UCHL1, PARK5, SNCA, NACP, PARKl, PARK4 , PRKN, PARK2, PDJ, DBH, NDUFV2, MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9, MECP2, RTT, PPMX, MRX16,MRX79, Acid Hydroxylase 2, Axonin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), CONT, DRD (Drdla), SLC6A, DAOA, DTNBP1, Dao (Daol), APH-l (α and β ), Presenilin (Psenl), Ncstn, PEN-2, Nosl, Parpl, Natl, Nat2, HTT, SBMA / SMAX1 / AR, FXN / X25, ATX3, TXN, ATXN2, DMPK, atrophin-1 , Atnl, CBP, VLDLR, Atxn7 and AtxnlO) and eye diseases and conditions (such as Aber, Ccl2, Cc2, cp (ceruloplasmin), Timp3, cathepsin-D, Vldlr, Ccr2, CRYAA, CRYA1, CRYBB2, CRYB2 , PITX3, BFSP2, CP49, CP47, CRYAA, CRYAI, PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM , MIP, AQPO, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA, CRYAI, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1 , APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3, CDG2, TACSTD2, TROP2, M1SI, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD, KERA, CNA2, MYOC, TIGR, GLCIA , JO AG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1BI, GLC3A, OPA1, NTG, NPG, CYP1BI, GLC3A, CRB1, RP12, CRX, CORD2, CRD, RPGRIPI, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4, GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3, ELOVL4, ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD and VMD2). ,

[0101] In some embodiments, the carrier or payload is or encodes a factor that affects cell differentiation. As non-limiting examples, expression of one or more of Oct4, Klf4, Sox2, c-Myc, L-Myc, dominant negative p53, Nanog, Glisl, Lin28, TFIID, mir-302 / 367, or other miRNAs may Turn the cells into induced pluripotent stem (iPS) cells.

[0102] In some embodiments, the carrier or payload is or encodes factors for transdifferentiating cells. Non-limiting examples of factors include: for cardiomyocytes, one or more of GATA4, Tbx5, Mef2C, Myocd, Hand2, SRF, Mespl, SMARCD3; for neural cells, Ascii, Nurrl, LmxlA, Bm2, Mytll, NeuroDl, FoxA2; and for hepatocytes, Hnf4a, Foxal, Foxa2 or Foxa3. Peptides, proteins and peptides

[0103] Initial constructs and reference constructs of the invention may comprise, be encoded as polypeptides, proteins or peptide carriers or payloads, or be combined therewith. As used herein, the term "polypeptide" generally refers to a polymer of amino acids linked by peptide bonds and encompasses "proteins" and "peptides." Polypeptides of the invention include all polypeptides, proteins and / or peptides known in the art. Non-limiting categories of polypeptides include antigens, antibodies, antibody fragments, cytokines, peptides, hormones, enzymes, oxidants, antioxidants, synthetic peptides, and chimeric peptides.

[0104] As used herein, the term "peptide" generally refers to shorter polypeptides of about 50 amino acids or less. Peptides with only two amino acids may be called "dipeptides." Peptides with only three amino acids may be called "tripeptides." Polypeptides generally refer to polypeptides having about 4 to about 50 amino acids. Peptides may be obtained by any method known to those skilled in the art. In some embodiments, the peptide can be expressed in culture. In some embodiments, peptides can be obtained via chemical synthesis (eg, solid phase peptide synthesis).

[0105] In some embodiments, the initial constructs and baseline constructs of the present invention may comprise, encode, or be combined with a carrier or payload that is a simple carrier or payload that yields amino acids and occasionally small carbohydrates upon hydrolysis. protein. Non-limiting examples of simple proteins include albumins, proteinoids, globulins, glutenins, histones, and protamines.

[0106] In some embodiments, the initial constructs and baseline constructs of the invention may comprise, encode, or be associated with a cargo or payload that is a bound protein, which may be a simple protein bound to a non-protein. . Non-limiting examples of binding proteins include glycoproteins, hemoglobin, lecithin, nucleoproteins, and phosphoproteins.

[0107] In some embodiments, the initial constructs and baseline constructs of the invention may comprise, encode, or be combined with a cargo or payload that is a derived protein that is obtained by chemical or physical means from a simple or Binding protein-derived proteins. Non-limiting examples of derivatized proteins include denatured proteins and peptides.

[0108] In some embodiments, the polypeptide, protein or peptide may be unmodified.

[0109] In some embodiments, a polypeptide, protein or peptide can be modified. Types of modifications include, but are not limited to, phosphorylation, glycosylation, acetylation, ubiquitination / ubiquitination, methylation, palmitrylation, quinone, amidation, myristoylation, pyrrolidone carboxylation Acid, hydroxylation, phosphopantetheine, isoprenylation, GPI anchoring, oxidation, ADP ribosylation, sulfation, S-nitrosylation, citrulline, nitration, γ-carboxylation Glutamic acid, methylation, putrescine, lysine, topaquinone (TPQ), bromide, lysine topaquinone (LTQ), tryptophan tryptophylquinone (TTQ) , iodinated and cysteine ​​tryptophan quinone (CTQ). In some aspects, a polypeptide, protein or peptide can be modified by subsequent transcriptional modifications that can affect its structure, subcellular localization and / or function.

[0110] In some embodiments, a polypeptide, protein or peptide can be modified using phosphorylation. Phosphorylation, or the addition of phosphate groups to serine, threonine, or tyrosine residues, is one of the most common forms of protein modification. Protein phosphorylation plays an important role in fine-tuning signaling in intracellular signaling cascades.

[0111] In some embodiments, a polypeptide, protein or peptide can be modified using ubiquitination, which is the covalent attachment of ubiquitin to a protein of interest. Ubiquitination-mediated protein turnover has been shown to play a role in driving the cell cycle as well as protein-degradation-independent intracellular signaling pathways.

[0112] In some embodiments, polypeptides, proteins or peptides can be modified using acetylation and methylation that can play a role in regulating gene expression. As non-limiting examples, acetylation and methylation can mediate the formation of chromosomal domains (such as euchromatin and heterochromatin), which may have an impact on mediating gene silencing.

[0113] In some embodiments, a polypeptide, protein or peptide can be modified using glycosylation. Glycosylation is one of the attachments of a large number of glycan groups and is a modification that occurs in approximately half of all proteins and plays a role in biological processes, including but not limited to embryonic development, cell division, and protein structure adjust. The two main types of protein glycosylation are N-glycosylation and O-glycosylation. For N-glycosylation, the glycans are linked to asparagine; and for O-glycosylation, the glycans are linked to serine or threonine.

[0114] In some embodiments, a polypeptide, protein or peptide can be modified using sumoylation. Sumoylation is the addition of SUMO (small ubiquitin-like modulator) to a protein and is a post-translational modification similar to ubiquitination. Antibody

[0115] As used herein, the term "antibody" is used in its broadest sense and specifically encompasses various embodiments, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., formed from at least two intact antibodies) bispecific antibodies) and antibody fragments (such as bifunctional antibodies), as long as they exhibit the required biological activity (such as "functionality"). Antibodies are primarily amino acid-based molecules, which are monomeric or multimeric polypeptides that contain at least one amino acid region derived from a known or parent antibody sequence and at least one amino acid region derived from a non-antibody sequence. Antibodies may contain one or more modifications (including, but not limited to, added sugar moieties, fluorescent moieties, chemical tags, etc.). For purposes herein, an "antibody" may include heavy and light chain variable domains and an Fc region.

[0116] The carrier or payload may comprise or may encode a polypeptide that forms one or more functional antibodies.

[0117] In some embodiments, the carrier or payload may comprise or may encode to form or act as any antibody, including but not limited to antibodies known in the art and / or may be marketed therapeutically, diagnostically, or for research purposes. Antibody peptides for sale. Additionally, the carrier or payload may comprise or may encode such antibodies or fragments of antibodies, such as, but not limited to, variable domains or complementarity determining regions (CDRs).

[0118] As used herein, the term "natural antibody" refers to a general heterotetraglycan protein of approximately 150,000 daltons consisting of two identical light (L) chains and two identical heavy (H) chains. The genes encoding antibody heavy and light chains are known and their respective constituent segments have been well characterized and described (Matsuda, F. et al., 1998. The Journal of Experimental Medicine. 188(11); 2151-62 and Li, A . et al., 2004. Blood. 103(12): 4602-9, the contents of each case are incorporated into this article by full-text citation). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds in the heavy chain of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain Comparison. As used herein, the term "light chain" refers to a component of an antibody from any vertebrate species designated as one of two distinct types based on the amino acid sequences of the constant domains, termed kappa and lambda. . Antibodies can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chain. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2 .

[0119] As used herein, the term "variable domain" refers to a specific antibody domain found on both the heavy and light chains of an antibody that widely differs in sequence among antibodies and is used for the binding of each specific antibody to its specific antigen. and specificity. The variable domain contains the hypervariable region. As used herein, the term "hypervariable region" refers to the region within a variable domain that contains the amino acid residues responsible for antigen binding. The amino acids present within the hypervariable region determine the structure of the complementarity-determining region (CDR) that becomes part of the antibody's antigen-binding site. As used herein, the term "CDR" refers to the region of an antibody that contains a structure complementary to its target antigen or epitope. The other parts of the variable domain that do not interact with the antigen are called framework (FW) regions. Antigen binding sites (also called antigen combination sites or paratopes) contain the amino acid residues necessary for interaction with a specific antigen. The exact residues that constitute the antigen-binding site are usually elucidated by cocrystallography with bound antigen, however, computational assessments can also be used based on comparisons with other antibodies (Strohl, W.R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Chapter 3, pages 47-54, the content of which is incorporated by reference in its entirety). Determining the residues that make up the CDR may include the use of numbering schemes, including but not limited to those taught by: Kabat [Wu, T.T. et al., 1970, JEM, 132(2):211-50 and Johnson, G. et al., 2000, Nucleic Acids Res. 28(1): 214-8, the contents of which are each incorporated by reference in their entirety], Chothia [Chothia and Lesk, J. Mol. Biol. 196, 901 (1987), Chothia et al. Human, Nature 342, 877 (1989) and Al-Lazikani, B. et al., 1997, J. Mol. Biol. 273(4):927-48, the contents of which are each incorporated by reference in their entirety], Lefranc (Lefranc, M.P. et al., 2005, Immunome Res. 1:3) and Honegger (Honegger, A. and Pluckthun, A. 2001. J. Mol. Biol. 309(3):657-70, the contents of which are respectively The full text is incorporated into this article by reference).

[0120] The V H and V L domains each have three CDRs. In order of appearance as they move along the variable domain polypeptide from the N-terminus to the C-terminus, the V LCDRs are referred to herein as CDR-L1, CDR-L2, and CDR-L3. In order of appearance as they move along the variable domain polypeptide from the N-terminus to the C-terminus, the V HCDRs are referred to herein as CDR-H1, CDR-H2, and CDR-H3. Each of the CDRs has an advantageous typical structure except CDR-H3, which contains amino acid sequences that can be highly variable in sequence and length between antibodies, thereby creating a variety of three-dimensional structures in the antigen-binding domain. In some cases, CDR-H3 can be analyzed within a panel of related antibodies to assess antibody diversity.

[0121] Various methods of determining CDR sequences are known in the art and can be applied to known antibody sequences. The system described by Kabat, also known as "according to Kabat numbering", "Kabat numbering", "Kabat definition" and "Kabat notation", provides an unambiguous residue numbering system applicable to any variable domain of an antibody and provides a way to define each chain The precise residue boundaries of the three CDRs. (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987) and (1991), the contents of which are incorporated by reference in their entirety). Kabat CDRs include approximately residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain, and residues 31-35 (CDR1) in the heavy chain variable domain , 50-65 (CDR2) and 95-102 (CDR3). Chothia and colleagues found that certain sub-portions within the Kabat CDR exhibit almost identical peptide backbone configurations despite being extremely diverse at the amino acid sequence level. (Chothia et al. (1987) J. Mol. Biol. 196: 901-917; and Chothia et al. (1989) Nature 342: 877-883, the contents of which are each incorporated herein by reference in their entirety). These CDRs may be referred to as "Chothia CDRs", "Chothia numbering" or "according to Chothia numbering" and approximately include residues 24-34 (CDR1), 50-56 (CDR2) and 89- in the light chain variable domain. 97 (CDR3), and 26-32 (CDR1), 52-56 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain. Mol. Biol. 196:901-917 (1987). The system described by MacCallum, also known as "according to MacCallum numbering" or "MacCallum numbering", consists of approximately residues 30-36 (CDR1), 46-55 (CDR2) and 89-96 (CDR3) in the light chain variable domain ) and residues 30-35 (CDR1), 47-58 (CDR2) and 93-101 (CDR3) in the heavy chain variable domain. (MacCallum et al. ((1996) J. Mol. Biol. 262(5):732-745), the contents of which are incorporated herein by reference in their entirety). The system described by AbM, also known as "numbering according to AbM" or "AbM numbering", consists of approximately residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain ) and residues 26-35 (CDR1), 50-58 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain. The IMGT (INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM) numbering of the variable region can also be used, which is the numbering of residues in the immunoglobulin variable heavy or light chain according to the IIMGT method (Lefranc, M.-P., "The IMGT" "unique numbering for immunoglobulins, T cell Receptors and Ig-like domains", The Immunologist, 7, 132-136 (1999), which is incorporated by reference in its entirety). As used herein, "IMGT sequence number" or "IMTG numbering" refers to the numbering of the sequence encoding the variable region according to IMGT. For heavy chain variable domains, when numbered according to IMGT, the hypervariable region ranges from amino acid positions 27 to 38 for CDR1, amino acid positions 56 to 65 for CDR2, and For CDR3, it is in the range of amino acid positions 105 to 117. For light chain variable domains, when numbered according to IMGT, the hypervariable region ranges from amino acid positions 27 to 38 for CDR1, amino acid positions 56 to 65 for CDR2, and For CDR3, it is in the range of amino acid positions 105 to 117.

[0122] In some embodiments, the carrier or payload may comprise or may encode antibodies that have been produced using methods known in the art, such as, but not limited to, immunization and presentation techniques (e.g., phage display, yeast display, and ribose display). In vivo presentation), fusion tumor technology, heavy chain and light chain variable region cDNA sequences selected from fusion tumors or other sources.

[0123] In some embodiments, the carrier or payload may comprise or may encode an antibody developed using any naturally occurring or synthetic antigen. As used herein, an "antigen" is an entity that induces or induces an immune response in an organism. An immune response is characterized by the response of an organism's cells, tissues and / or organs to the presence of foreign entities. Such an immune response typically causes the organism to produce one or more antibodies directed against a foreign entity, such as an antigen or a portion of an antigen. As used herein, "antigen" also refers to a binding partner of a specific antibody or binding agent in a presentation library.

[0124] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of cells (or pure lines), that is, except for possible variants that may arise during the production of the monoclonal antibody (such Variants usually exist in small amounts), the individual antibodies that make up the population are identical and / or bind the same epitope. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0125] The modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. Monoclonal antibodies as used herein include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or identical to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass. source, and the remainder of the chain(s) is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.

[0126] As used herein, the term "humanized antibody" refers to a chimeric antibody that contains a minimal portion from one or more non-human (e.g., murine) antibody sources and the remaining portion is derived from one or more human immune sources. Source of globulin. Humanized antibodies are, to a large extent, human immunoglobulins (receptor antibodies) in which the residues of the hypervariable region of the antibody from the receptor have been derived from a non-human species (such as mouse, rat, rabbit or non-human Residue substitutions in the hypervariable region of an antibody (donor antibody) with the desired specificity, affinity and / or ability of a primate.

[0127] In some embodiments, the carrier or payload may comprise or encode an antibody mimetic. As used herein, the term "antibody mimetic" refers to any molecule that mimics the function or action of an antibody and binds specifically and with high affinity to its molecular target. In some embodiments, the antibody mimetic can be a monofunctional antibody designed to incorporate the fibronectin type III domain (Fn3) as the protein backbone. In some embodiments, the antibody mimetic can be an antibody mimetic known in the art, including but not limited to affinity antibody molecules, affilins, avidin, ananti-carbin, high-affinity Multimers, Centyrins, DARPINS™, fynomers, Kunitz domains and domain peptides. In other embodiments, an antibody mimetic may include one or more non-peptide regions. [Antibody fragments and variants]

[0128] In some embodiments, the carrier or payload may comprise or may encode an antibody fragment comprising an antigen-binding region from a full-length antibody. Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site. Residual "Fc" fragments are also produced, whose name reflects their ability to crystallize easily. Pepsin treatment produces F(ab') 2 fragments, which have two antigen-binding sites and are still able to cross-link antigen. Compounds and / or compositions of the invention may contain one or more of these fragments.

[0129] In some embodiments, the Fc region can be a modified Fc region, wherein the Fc region can have a single amino acid substitution compared to the corresponding sequence of a wild-type Fc region, wherein the single amino acid substitution results in a wild-type Fc region. Fc region having better properties than those of the Fc region. Non-limiting examples of Fc properties that can be altered by single amino acid substitutions include binding properties or response to pH conditions.

[0130] As used herein, the term "Fv" refers to an antibody fragment that contains the smallest fragment required to form a complete antigen-binding site on the antibody. These regions consist of dimers of a heavy chain and a light chain variable domain that are tightly, non-covalently associated. Fv fragments can be produced by proteolytic cleavage, but most are unstable. Recombinant methods for generating stable Fv fragments are known in the art, typically via insertion of a flexible linker between the light chain variable domain and the heavy chain variable domain to form a single chain Fv (scFv) or via the insertion of a heavy chain variable domain into the heavy chain Fv. A disulfide bridge is introduced between the chain variable domain and the light chain variable domain.

[0131] As used herein, the term "single chain Fv" or "scFv" refers to a fusion protein of VH and VL antibody domains in which these domains are linked together into a single polypeptide chain by a flexible peptide linker. In some embodiments, the Fv polypeptide linker enables the scFv to form the structure required for antigen binding. In some embodiments, scFvs are used in conjunction with phage display, yeast display, or other presentation methods, where they can be expressed in association with surface members (e.g., phage coat proteins) and used to identify high-affinity peptides for a given antigen.

[0132] As used herein, the term "antibody variant" refers to a modified antibody (relative to a native or starting antibody) or biomolecule (e.g., an antibody mimetic) that is structurally and / or functionally similar to the native or starting antibody. Antibody variants may differ in their amino acid sequence, composition or structure compared to the native antibody. Antibody variants may include, but are not limited to, antibodies with altered isotypes (e.g., IgA, IgD, IgE, IgG 1, IgG 2, IgG 3, IgG 4, or IgM), humanized variants, optimized variants, multispecific Specific antibody variants (e.g., bispecific variants) and antibody fragments. [Multispecific antibodies]

[0133] In some embodiments, the carrier or payload may be or encode an antibody that binds more than one epitope. As used herein, the term "multifunctional antibody" or "multispecific antibody" refers to an antibody in which two or more variable regions bind to different epitopes. The epitopes can be on the same or different targets. In certain embodiments, multispecific antibodies are "bispecific antibodies" that recognize two different epitopes on the same or different antigens.

[0134] In some embodiments, multispecific antibodies can be prepared by methods used by BIOATLA® and described in International Patent Publication WO201109726, the contents of which are incorporated herein by reference in their entirety. First, a library of homogeneous, naturally occurring antibodies (i.e., mammalian cell surface-presented) is generated by any method known in the art, followed by FACSAria or another screening method targeting two or more Multispecific antibodies that specifically bind to the target antigen are screened. In some embodiments, the identified multispecific antibodies are further evolved by any method known in the art to generate a set of modified multispecific antibodies. The modified multispecific antibodies are screened for binding to the target antigen. In some embodiments, multispecific antibodies can be further optimized by screening evolved modified multispecific antibodies for optimization or desired characteristics.

[0135] In some embodiments, multispecific antibodies can be prepared by methods used by BIOATLA® and described in U.S. Publication No. US20150252119, the contents of which are incorporated herein by reference in their entirety. In one approach, the variable domains of two parent antibodies (where the parent antibodies are monoclonal antibodies) are aligned so that a single light chain is functionally identical to that of the two different parent antibodies, using any method known in the art. Heavy chains evolve in a complementary manner. Another approach requires evolving the heavy chain of a single parent antibody to recognize a second target antigen. A third approach involves evolving the light chain of the parent antibody to recognize a second target antigen. Methods for polypeptide evolution are described in International Publication WO2012009026, the contents of which are incorporated herein by reference in their entirety and, as non-limiting examples, include Comprehensive Positional Evolution (CPE), Combinatorial Protein Synthesis (CPS), Comprehensive Positional Evolution (CPS), insertion (CPI), comprehensive positional deletion (CPD), or any combination thereof. The Fc region of the multispecific antibody described in US Publication No. US20150252119 can be generated using the knob-in-hole approach or any other method that enables the Fc domain to form heterodimers. The resulting multispecific antibodies can be further evolved to obtain improved characteristics or properties, such as binding affinity for the target antigen. [Bispecific antibody]

[0136] In some embodiments, the carrier or payload may be or encode a bispecific antibody. As used herein, the term "bispecific antibody" refers to an antibody capable of binding two different antigens. Such antibodies typically contain regions from at least two different antibodies. Such antibodies typically contain antigen-binding regions from at least two different antibodies. For example, bispecific monoclonal antibodies (BsMAb, BsAb) are artificial proteins composed of fragments of two different monoclonal antibodies, thus enabling the BsAb to bind to two different types of antigens.

[0137] In some cases, the carrier or payload may be or encode a bispecific antibody comprising antigen-binding regions from two different anti-tau antibodies. For example, such bispecific antibodies can comprise binding regions from two different antibodies.

[0138] Bispecific antibody frameworks may include any of those described in: Riethmuller, G., 2012. Cancer Immunity. 12:12-18; Marvin, J.S. et al., 2005. Acta Pharmacologica Sinica. 26( 6):649-58; and Schaefer, W. et al., 2011. PNAS. 108(27):11187-92, the respective contents of which are incorporated by reference in their entirety.

[0139] A new generation of BsMAb has been developed, called a "trifunctional bispecific" antibody. These antibodies are composed of two heavy chains and two light chains, each derived from two different antibodies, with two Fab regions (arms) targeting two antigens, and the Fc region (feet) containing the two heavy chains and forming a third binding site.

[0140] Of the two paratopes forming the top of the variable domain of a bispecific antibody, one can be directed to the target antigen and the other to a T lymphocyte antigen, such as CD3. In the case of trifunctional antibodies, the Fc region can additionally bind to cells expressing Fc receptors, such as macrophages, natural killer (NK) cells, or dendritic cells. In summary, the targeted cell connects with one or two cells in the immune system, which then destroys the targeted cell.

[0141] Other types of bispecific antibodies have been designed to overcome certain problems, such as short half-life, immunogenicity, and side effects caused by interleukin release. It includes chemically linked Fabs (composed only of the Fab region), various types of bivalent and trivalent single-chain variable fragments (scFv), and fusion proteins that mimic the variable domains of two antibodies. The most recently developed of these newer formats are bispecific T cell engager molecules (BiTEs) and mAb2, antibodies engineered to contain Fcab antigen-binding fragments in place of the Fc constant region.

[0142] Using molecular genetics, two scFvs can be tandem engineered into a single polypeptide separated by a linker domain, termed a "tandem scFv" (tascFv). TascFv has been found to be poorly soluble and requires refolding when produced in bacteria, or it can be produced in mammalian cell culture systems, thereby avoiding the need for refolding but potentially resulting in poor yields. The construction of a tascFv with genes for two different scFvs results in a "dual-specific single-chain variable fragment" (dual-scFv). Commercial companies have only developed two tascFvs clinically; both are bispecific agents for tumor indications actively developed by Micromet in the early stages, and are described as "bispecific T cell engager molecules (BiTE)." Blinatumomab is an anti-CD19 / anti-CD3 bispecific tascFv that enhances T cell responses in stage 2 B-cell non-Hodgkin's lymphoma. MT110 is an anti-EP-CAM / anti-CD3 bispecific tascFv that enhances T cell responses against stage 1 solid tumors. Affimed is also studying bispecific quadrivalent "TandAb".

[0143] In some embodiments, the carrier or payload may be or encode an antibody comprising a single antigen binding domain. These molecules are extremely small, with molecular weights approximately one-tenth that observed for full-size mAbs. Other antibodies may include "nanobodies" derived from the antigen-binding variable heavy chain region (V HH) of heavy chain antibodies found in camels and llamas, which do not have light chains.

[0144] Memorial Sloan-Kettering Cancer Center's PCT Publication WO2014144573, the contents of which are incorporated herein by reference in its entirety, discloses and advocates for use in the preparation of two compounds with improved properties relative to multispecific binding agents that do not possess dimerization capabilities. Polymeric technology for polymultispecific binding agents, such as fusion proteins containing antibody components.

[0145] In some cases, the carrier or payload may be or encode a tetravalent bispecific antibody (TetBiAb, as disclosed and claimed in PCT Publication WO2014144357, the contents of which are incorporated herein in its entirety). TetBiAb features a second pair of Fab fragments with a second antigen specificity linked to the C-terminus of the antibody, thus providing a bivalent molecule for each of the two antigen specificities. Tetravalent antibodies are produced through genetic engineering by covalently linking the antibody heavy chain to the Fab light chain (which binds to its homologous, co-expressing Fab heavy chain).

[0146] In some aspects, the carrier or payload may be or encode a biosynthetic antibody, as described in U.S. Patent No. 5,091,513 (the contents of which are incorporated herein by reference in their entirety). Such antibodies may include one or more sequences of amino acids that constitute a region that serves as a biosynthetic antibody binding site (BABS). Sites include 1) non-covalently or disulfide-bonded synthetic VH and VL dimers, 2) VH-VL or VL-VH single chains where VH and VL are connected by a polypeptide linker, or 3) individual V H or V L domains. The binding domain includes linked CDR and FR regions, which can be derived from separate immunoglobulins. Biosynthetic antibodies may also include other polypeptide sequences that serve, for example, as enzymes, toxins, binding sites, or attachment sites to fixed media or radioactive atoms. Methods for making biosynthetic antibodies, for designing BABS with any specificity that can be elicited by in vivo production of antibodies, and for making analogs thereof are disclosed.

[0147] In some embodiments, the carrier or payload may be or encode an antibody having the antibody receptor framework taught in U.S. Patent No. 8,399,625. Such antibody receptor frameworks may be particularly suitable for accepting CDRs from antibodies of interest. In some cases, CDRs from anti-tau antibodies known in the art or developed according to the methods presented herein can be used. [Miniaturized antibodies]

[0148] In some embodiments, the carrier or payload may be or encode a "miniaturized" antibody. Best examples of mAb miniaturization include the Small Modular ImmunoPharmaceuticals (SMIP) from Trubion Pharmaceuticals. These molecules (which can be monovalent or bivalent) are recombinant single-chain molecules containing a VL, a VH antigen-binding domain, and one or two constant "effector" domains (all connected by a linker domain). It is possible that such molecules could provide the enhanced tissue or tumor penetration conferred by the fragments while retaining the advantages of the immune effector functions conferred by the constant domains. At least three "miniaturized" SMIPs have entered clinical development. TRU-015, an anti-CD20 SMIP developed in partnership with Wyeth, is a state-of-the-art program in Phase 2 development for rheumatoid arthritis (RA). Earlier attempts in systemic lupus erythematosus (SLE) and B-cell lymphoma were ultimately discontinued. Trubion and Facet Biotechnology are collaborating to develop TRU-016, an anti-CD37 SMIP for the treatment of CLL and other lymphomas, which has reached Phase 2. Wyeth has approved the anti-CD20 SMIP SBI-087 for the treatment of autoimmune diseases, including RA, SLE and possibly multiple sclerosis, but these programs are still in the earliest stages of clinical testing. [Bifunctional antibody]

[0149] In some embodiments, the carrier or payload may be or encode a bifunctional antibody. As used herein, the term "diabody" refers to a small antibody fragment with two antigen-binding sites. Diabodies comprise a heavy chain variable domain, VH, linked to a light chain variable domain, VL, in the same polypeptide chain. By using a linker that is too short to allow pairing between two domains on the same chain, the domain is forced to pair with a complementary domain on the other chain and creates two antigen-binding sites.

[0150] Bifunctional antibodies are functional bispecific single chain antibodies (bscAb). These bivalent antigen-binding molecules are composed of non-covalent dimers of scFv and can be produced in mammalian cells using recombinant methods. (See, eg, Mack et al., Proc. Natl. Acad. Sci., 92: 7021-7025, 1995). Few bifunctional antibodies have entered clinical development. In a study sponsored by the Beckman Research Institute of the City of Hope (Clinicaltrials.gov NCT00647153), an iodine-123-labeled bifunctional antibody version of the anti-CEA chimeric antibody cT84.66 was evaluated for preoperative immunization against colorectal cancer. Flicker scan detection. [Single antibody]

[0151] In some embodiments, the carrier or payload may be or encode a "unibody" in which the hinge region has been removed from the IgG4 molecule. Although IgG4 molecules are unstable and can exchange light chain-heavy chain heterodimers with each other, deletion of the hinge region completely prevents heavy chain-heavy chain pairing, leaving highly specific monovalent light chain / heavy chain heterodimers. The Fc region is also preserved to ensure stability and in vivo half-life. This configuration minimizes the risk of immune activation or oncogenic growth because IgG4 interacts poorly with FcR and monovalent monoantibodies are unable to promote intracellular signaling complex formation. However, this view is mainly supported by laboratory rather than clinical evidence. Other antibodies can be "miniaturized" antibodies, which are compact 100 kDa antibodies. [Intracellular antibodies]

[0152] In some embodiments, the carrier or payload may be or encode an intrabody. The term "intrabody" refers to a form of an antibody that is not secreted by the cell in which it is produced, but instead targets one or more intracellular proteins. Intrabodies can be used to affect multiple cellular processes, including but not limited to intracellular migration, transcription, translation, metabolic processes, proliferative signaling, and cell division. In some embodiments, methods of the invention may include intrabody-based therapies. In some such embodiments, the variable domain sequences and / or CDR sequences disclosed herein can be incorporated into one or more constructs for intrabody-based therapies. For example, an intrabody may target one or more glycated intracellular proteins or may modulate the interaction between one or more glycated intracellular proteins and an alternative protein.

[0153] Intrabodies against intracellular targets were first described more than two decades ago (Biocca, Neuberger and Cattaneo EMBO J. 9: 101-108, 1990, the contents of which are incorporated herein by reference in their entirety). Intracellular expression of intrabodies in different compartments of mammalian cells allows blocking or modulating the function of endogenous molecules (Biocca et al., EMBO J. 9: 101-108, 1990; Colby et al., Proc. Natl. Acad. Sci. U.S.A. 101: 17616-21, 2004, the contents of which are incorporated herein by reference in their entirety). Intrabodies can alter protein folding, protein-protein, protein-DNA, protein-RNA interactions, and protein modifications. It can induce phenotypic gene knockout and act as a neutralizing agent by directly binding to the target antigen, by diverting its intracellular migration, or by inhibiting its binding to binding partners. They are primarily used as research tools and emerge as therapeutic molecules for the treatment of human diseases such as viral pathologies, cancer and misfolding diseases. Regarding their use in therapy, the rapidly growing biological market for recombinant antibodies provides intrabodies with enhanced binding specificity, stability and solubility as well as lower immunogenicity.

[0154] In some embodiments, intrabodies are preferred over interfering RNA (iRNA); for example, iRNAs have been shown to exert a variety of non-specific effects, whereas intrabodies have been shown to have high specificity and affinity for target antigens. Furthermore, as proteins, intrabodies have a much longer active half-life than iRNA. Therefore, when the active half-life of the intracellular target molecule is long, the process of gene silencing through iRNA may be slower, while the effect of intracellular antibodies may be almost instantaneous. Ultimately, it may be possible to design intrabodies to block certain binding interactions with specific target molecules while avoiding other effects.

[0155] Intrabodies are typically single-chain variable fragments (scFv) expressed by recombinant nucleic acid molecules and engineered to persist within the cell (eg, in the cytoplasm, endoplasmic reticulum, or extracellular cytoplasm). Intrabodies can be used, for example, to eliminate the function of proteins to which the intrabodies bind. The expression of intrabodies can also be modulated by using an inducible promoter in a nucleic acid expression vector containing the intrabodies. Intrabodies for use in the viral genomes of the invention can be generated using methods known in the art, such as those disclosed and reviewed in: Marasco et al., 1993 Proc. Natl. Acad. Sci. USA, 90: 7889-7893;Chen et al., 1994, Hum. Gene Ther.5:595-601;Chen et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 5932-5936;Maciejewski et al. , 1995, Nature Med., 1: 667-673;Marasco, 1995, Immunotech, 1: 1-19;Mhashilkar et al., 1995, EMBO J. 14: 1542-51;Chen et al., 1996, Hum. Gene Therap ., 7: 1515-1525;Marasco, Gene Ther. 4:11-15, 1997;Rondon and Marasco, 1997, Annu. Rev. Microbiol.51:257-283;Cohen et al., 1998, Oncogene17:2445-56 ;Proba et al., 1998, J. Mol. Biol. 275:245-253;Cohen et al., 1998, Oncogene17:2445-2456;Hassanzadeh et al., 1998, FEBS Lett. 437:81-6;Richardson et al., 1998, Gene Ther. 5:635-44; Ohage and Steipe, 1999, J. Mol. Biol. 291:1119-1128; Ohage et al., 1999, J. Mol. Biol. 291:1129-1134; Wirtz and Steipe , 1999, Protein Sci. 8:2245-2250; Zhu et al., 1999, J. Immunol. Methods231:207-222; Arafat et al., 2000, Cancer Gene Ther. 7:1250-6; der Maur et al., 2002 , J. Biol. Chem. 277:45075-85; Mhashilkar et al., 2002, Gene Ther. 9:307-19; and Wheeler et al., 2003, FASEB J. 17: 1733-5; and references cited therein ). In particular, CCR5 intrabodies have been generated by Steinberger et al., 2000, Proc. Natl. Acad. Sci. USA 97:805-810). See generally Marasco, WA, 1998, "Intrabodies: Basic Research and Clinical Gene Therapy Applications" Springer: New York; and for a review of scFv, see Pluckthun "The Pharmacology of Monoclonal Antibodies", 1994, vol. 113, Rosenburg and Moore eds . Springer-Verlag, New York, pp. 269-315; the contents of which are each incorporated by reference in their entirety.

[0156] Sequences from donor antibodies can be used to generate intrabodies. Intrabodies are typically expressed recombinantly within cells as single domain fragments (such as isolated V H and V L domains) or single chain variable fragment (scFv) antibodies. For example, intrabodies are typically expressed as a single polypeptide to form a single chain antibody comprising variable domains of heavy and light chains joined by a flexible linker polypeptide. Intrabodies typically lack disulfide bonds and are capable of modulating the expression or activity of target genes via their specific binding activity. Single-chain antibodies can also be expressed as single-chain variable region fragments joined to light chain constant regions.

[0157] As is known in the art, intrabodies can be engineered into recombinant polynucleotide vectors to encode daughter cell migration signals at their N or C terminus to allow expression at high concentrations in the daughter cell compartment where the target protein is located. . For example, intrabodies targeting the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and optionally a C-terminal ER retention signal. Intrabodies intended to be active in the nucleus are engineered to include a nuclear localization signal. The lipid moiety is conjugated to the intrabody to tether the intrabody to the cytoplasmic side of the plasma membrane. Intrabodies can also be targeted to function in the cytosol. For example, cytosolic intrabodies are used to sequester factors within the cytosol, thereby preventing their transport to their natural cellular destination.

[0158] There are certain technical challenges regarding the expression of intrabodies. Specifically, the protein conformational folding and structural stability of newly synthesized intracellular antibodies in cells are affected by the reducing conditions of the intracellular environment.

[0159] The intrabodies of the invention are due to their almost unlimited ability to specifically recognize different conformations of proteins, including pathological isoforms, and because they can target potential aggregation sites (both intracellular and extracellular sites) It may be a promising treatment for misfolding diseases, including tauopathies, prion diseases, Alzheimer's, Parkinson's and Huntington's. agent. These molecules may act as neutralizers for amyloidogenic proteins by preventing amyloidogenic proteins from aggregating, and / or act as molecular schedulers of intracellular traffic by causing proteins to reroute from their potential aggregation sites. [Maximum Antibody]

[0160] In some embodiments, the carrier or payload may be or encode a maxibody (a bivalent scFV fused to the amino terminus of the Fc (CH2-CH3 domain) of an IgG). Chimeric Antigen Receptor (CAR)

[0161] In some embodiments, the carrier or payload may be or encode a chimeric antigen receptor (CAR), which when transduced into immune cells, such as T cells and NK cells, can redirect immune cells to target Targets (eg, tumor cells) that represent molecules recognized by the extracellular target portion of the CAR.

[0162] As used herein, the term "chimeric antigen receptor (CAR)" refers to a synthetic receptor that mimics the TCR on the surface of T cells. Generally speaking, CAR consists of an extracellular targeting domain, a transmembrane domain / region, and an intracellular signaling / activation domain. In a standard CAR receptor, the components: extracellular targeting domain, transmembrane domain and intracellular signaling / activation domain are linearly constructed into a single fusion protein. The extracellular region contains targeting domains / portions (eg scFv) that recognize specific tumor antigens or other tumor cell surface molecules. The intracellular domain may contain the signaling domain of the TCR complex (eg, the signaling domain of CD3ζ) and / or one or more costimulatory signaling domains, such as those from CD28, 4-1BB (CD137), and OX-40 (CD134) Those who. For example, "first-generation CAR" only has a CD3 ζ signaling domain, and in order to increase T cell persistence and proliferation, a costimulatory intracellular domain is added, resulting in a CD3 ζ signaling domain plus a costimulatory signaling domain. second-generation CARs, and third-generation CARs with a CD3 ζ signaling domain plus two or more costimulatory signaling domains. When expressed by a T cell, a CAR confers on the T cell an antigen specificity determined by the extracellular targeting portion of the CAR. In some aspects, one or more elements, such as homing and suicide genes, can be added to create more competent and safer CAR architectures (so-called fourth-generation CARs).

[0163] In some embodiments, the extracellular targeting domain is joined to the intracellular signaling domain via a hinge (also known as a spacer domain or spacer) and a transmembrane region. The hinge connects the extracellular targeting domain to a transmembrane domain that crosses the cell membrane and connects to the intracellular signaling domain. Due to the size of the target protein bound by the targeting moiety and the size and affinity of the targeting domain itself, changes in the hinge may be required to optimize the efficacy of CAR-transformed cells against cancer cells. After the targeting moiety recognizes and binds to the target cell, the intracellular signaling domain generates an activation signal for the CAR T cell, which is further amplified by a "second signal" from one or more intracellular costimulatory domains. Once activated, CAR T cells can destroy target cells.

[0164] In some embodiments, a CAR can be divided into two parts, with each part linked to a dimerization domain such that input that triggers dimerization promotes the assembly of a fully functional receptor. Wu and Lim reported a split CAR in which the extracellular CD19-binding domain and intracellular signaling elements were isolated and linked to the heterodimeric FKBP domain and FRB* in the presence of the rapamycin analog AP21967 (FKBP-rapamycin protein-binding T2089L mutant) domain. The split receptor assembles in the presence of AP21967 and activates T cells along with specific antigen binding (Wu et al., Science, 2015, 625(6258): aab4077, the contents of which are incorporated by reference in their entirety).

[0165] In some embodiments, the CAR can be designed as an inducible CAR that has incorporated the Tet-On inducible system into a CD19 CAR construct. CD19 CAR is only activated in the presence of doxycycline (Dox). Sakemura reported that compared with conventional CD19CAR T cells, Tet-CD19CAR T cells have equivalent cytotoxicity to CD19+ cell lines in the presence of Dox, and have equivalent interleukin production and proliferation after CD19 stimulation (Sakemura et al. , Cancer Immuno. Res., June 21, 2016, electronic version; its contents are incorporated into this article by reference in full). The dual system provides more flexibility for turning CAR expression on and off in transduced T cells.

[0166] In some embodiments, the carrier or payload may be or encode a first generation CAR, or a second generation CAR, or a third generation CAR, or a fourth generation CAR. In some embodiments, the carrier or payload may be or encode a complete CAR construct consisting of the extracellular domain, the hinge and transmembrane domains, and the intracellular signaling region. In other embodiments, the carrier or payload may be or encode components of a complete CAR construct, including an extracellular targeting moiety, a hinge region, a transmembrane domain, an intracellular signaling domain, one or more costimulatory domain and other additional components that improve the architecture and functionality of the CAR, including but not limited to leader sequences, homing components and safety switches or combinations of such components.

[0167] In some embodiments, the payload or payload may be or encode a tunable CAR. The reversible on-off switching mechanism allows management of acute toxicity caused by excessive CAR-T cell expansion. Ligand-conferred CAR regulation may effectively counteract tumor escape induced by antigen deletion, thereby avoiding functional exhaustion due to tonic signaling due to long-term antigen exposure and improving the persistence of CAR-expressing cells in vivo. Tunable CARs can be used to downregulate CAR performance with the goal of limiting tissue toxicity caused by tumor lysis syndrome. Down-regulation of CAR expression following anti-tumor efficacy prevents (1) off-target tumor toxicity caused by antigen expression in normal tissues; (2) antigen-independent activation in vivo. [Extracellular targeting domain] [ / ] [part]

[0168] In some embodiments, the extracellular target portion of the CAR can be any agent that recognizes and binds with high specificity and affinity to a given target molecule (eg, a neoantigen on a tumor cell). The target moiety can be: an antibody and its variants that specifically bind to the target molecule on the tumor cell; or an aptamer selected from a random sequence pool based on its ability to bind to the target molecule on the tumor cell; or can be combined with the target molecule on the tumor cell. Variants or fragments thereof that bind target molecules; or antigen recognition domains from native T-cell receptors (TCRs) (such as the extracellular domain of CD4 that recognizes HIV-infected cells); or foreign recognition components, such as those that cause recognition of carrier cell mediators. Interleukins that bind to target cells of receptors or natural ligands of receptors.

[0169] In some embodiments, the targeting domain of the CAR can be Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single chain variable fragment (scFv), Bi-scFv, (scFv)2, minibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, disulfide-stabilized Fv protein (dsFv), monofunctional antibody, nanobody, or derived from a specific recognition target The antigen-binding region of an antibody such as a tumor-specific antigen (TSA). In one embodiment, the targeting moiety is a scFv antibody. When expressed on the surface of CAR T cells and subsequently bound to target proteins on cancer cells, the scFv domain is able to maintain CAR T cell proximity to cancer cells and trigger T cell activation. scFv can be produced using conventional recombinant DNA technology techniques and is discussed herein.

[0170] In some embodiments, the targeting portion of the CAR construct can be an aptamer, such as a peptide aptamer, that specifically binds to the target molecule of interest. Peptide aptamers can be selected from a random sequence pool based on their ability to bind to the target molecule of interest.

[0171] In some embodiments, the targeting portion of the CAR construct can be a native ligand of the target molecule or a variant and / or fragment thereof capable of binding the target molecule. In some aspects, the targeting portion of the CAR can be a receptor for the target molecule. For example, full-length human CD27, as a CD70 receptor, can be fused in-frame with the signaling domain of CD3ζ, thereby forming a CD70-positive malignant CD27 chimeric receptors as immunotherapeutic agents for disease.

[0172] In some embodiments, the targeting portion of the CAR recognizes a tumor-specific antigen (TSA), such as a cancer neoantigen that is restricted to tumor cells.

[0173] As a non-limiting example, the CAR of the invention may comprise an extracellular targeting domain capable of binding to a tumor-specific antigen selected from: 5T4, 707-AP, A33, AFP (alpha-fetoprotein), AKAP-4 ( Kinase-anchored protein 4), ALK, α5β1-integrin, androgen receptor, Annexin II, α-actinin-4, ART-4, B1, B7H3, B7H4, BAGE (B melanoma antigen) , BCMA, BCR-ABL fusion protein, β-catenin, BKT-antigen, BTAA, CA-I (carbonic anhydrase I), CA50 (cancer antigen 50), CA125, CA15-3, CA195, CA242, calcium reticulum Protein, CAIX (carbonic anhydrase), CAMEL (cytotoxic T lymphocyte recognition antigen on melanoma), CAM43, CAP-1, apoptotic protease-8 / m, CD4, CD5, CD7, CD19, CD20, CD22, CD23, CD25, CD27 / m, CD28, CD30, CD33, CD34, CD36, CD38, CD40 / CD154, CD41, CD44v6, CD44v7 / 8, CD45, CD49f, CD56, CD68\KP1, CD74, CD79a / CD79b, CD103, CD123, CD133, CD138, CD171, cdc27 / m, CDK4 (cyclin-dependent kinase 4), CDKN2A, CDS, CEA (carcinoembryonic antigen), CEACAM5, CEACAM6, chromogranin, c-Met, c-Myc, coa - 1. CSap, CT7, CT10, cyclophilin B, cyclin B1, cytoplasmic tyrosine kinase cytokeratin, DAM-10, DAM-6, dek-can fusion protein, desmin, DEPDC1 (including DEP domain 1), E2A-PRL, EBNA, EGF-R (epidermal growth factor receptor), EGP-1 (epithelial glycoprotein-1) (TROP-2), EGP-2, EGP-40, EGFR (epidermal growth factor receptor receptor), EGFRvIII, EF-2, ELF2M, EMMPRIN, EpCAM (epithelial cell adhesion molecule), EphA2, Epstein-Barr virus antigen, Erb (ErbB1; ErbB3; ErbB4), ETA (epithelial tumor antigen), ETV6- AML1 fusion protein, FAP (fibroblast activation protein), FBP (folate binding protein), FGF-5, folate receptor, FOS-related antigen 1, fucosyl GM1, G250, GAGE ​​(GAGE-1; GAGE-2 ), galectin, GD2 (ganglioside), GD3, GFAP (glial fibrillary acidic protein), GM2 (carcinoembryonic antigen immunogenicity-1; OFA-I-1), GnT-V, Gp100 , H4-RET, HAGE (helicase antigen), HER-2 / neu, HIF (hypoxia-inducible factor), HIF-1, HIF-2, HLA-A2, HLA-A*0201-R170I, HLA-Al l, HMWMAA, Hom / Mel-40, HSP70-2M (heat shock protein 70), HST-2, HTgp-175, hTERT (or hTRT), human papilloma virus-E6 / human papilloma virus-E7 and E6, iCE (immune capture EIA), IGF-1R, IGH-IGK, IL-2R, IL-5, ILK (integrin-linked kinase), IMP3 (insulin-like growth factor II mRNA binding protein 3), IRF4 (interferon Regulator 4), KDR (kinase insertion domain receptor), KIAA0205, KRAB zinc finger protein (KID)-3; KID31, KSA (17-1A), K-ras, LAGE, LCK, LDLR / FUT (LDLR-Rock Fucosyltransferase AS fusion protein), LeY (Lewis Y), MAD-CT-1, MAGE (tyrosinase. Melanoma-associated antigen) (MAGE-1; MAGE-3), melanin-A tumor antigen (MART), MART-2 / Ski, MC1R (melanocortinogen 1 receptor), MDM2, mesothelin, MPHOSPH1, MSA (muscle-specific actin), mTOR (mammalian target of rapamycin), MUC-1, MUC-2, MUM-1 (melanoma-associated antigen (mutated) 1), MUM-2, MUM- 3. Myosin / m, MYL-RAR, NA88-A, N-acetylglucose transferase, neo-PAP, NF-KB (nuclear factor-κB), neurofilament, NSE (neuron-specific enol enzyme), Notch receptor, NuMa, N-Ras, NY-BR-1, NY-CO-1, NY-ESO-1, Oncostatin M, OS-9, OY-TES1, p53 mutant, p190 mini bcr-abl, pl5(58), pl85erbB2, pl80erbB-3, PAGE (prostate-related gene), PAP (prostatic acid phosphatase), PAX3, PAX5, PDGFR (platelet-derived growth factor receptor), involving piperidine and pyrrolidine Utilizing (PIPA) cytochrome P450, Pml-RAR alpha fusion protein, PR-3 (protease 3), PSA (prostate specific antigen), PSM, PSMA (prostatic stem cell antigen), PRAME (prior expression antigen of melanoma) , PTPRK, RAGE (renal tumor antigen), Raf (A-Raf, B-Raf and C-Raf), Ras, receptor tyrosine kinase, RCAS1, RGSS, ROR1 (receptor tyrosine kinase-like orphan receptor 1), RU1, RU2, SAGE, SART-1, SART-3, SCP-1, SDCCAG16, SP-17 (sperm protein 17), src-family, SSX (synovial sarcoma X breakpoint)-1, SSX- 2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, STAT-3, STAT-5, STAT-6, STEAD, STn survivin, syk-ZAP70, TA-90 (Mac-2 binding protein\cyclophilin C-related protein), TAAL6, TACSTD1 (tumor-associated calcium signal transducer 1), TACSTD2, TAG-72-4, TAGE, TARP (T cell receptor gamma alternative reading frame protein), TEL / AML1 fusion protein, TEM1, TEM8 (endosialin or CD248), TGFβ, TIE2, TLP, TMPRSS2 ETS fusion gene, TNF receptor (TNF-α receptor, TNF-β receptor; or TNF-γ receptor ), transferrin receptor, TPS, TRP-1 (tyrosine-related protein 1), TRP-2, TRP-2 / INT2, TSP-180, VEGF receptor, WNT, WT-1 (Wilm's tumor antigens) and XAGE.

[0174] In some embodiments, the carrier or payload may be or may encode a CAR comprising a universal immune receptor having a targeting moiety capable of binding to a labeled antigen.

[0175] In some embodiments, the carrier or payload may be or encode a CAR comprising a targeting moiety capable of binding to a pathogen antigen.

[0176] In some embodiments, the cargo or payload may be or encode a CAR that includes a targeting moiety capable of binding to non-protein molecules, such as tumor-associated glycolipids and carbohydrates.

[0177] In some embodiments, the carrier or payload may be or encode a CAR that includes a targeting moiety capable of binding to components within the tumor microenvironment, including proteins expressed in various tumor stromal cells, These tumor stromal cells include tumor-associated macrophages (TAMs), immature monocytes, immature dendritic cells, immunosuppressive CD4+CD25+ regulatory T cells (Tregs) and MDSCs.

[0178] In some embodiments, the carrier or payload may be or encode a CAR that includes a targeting portion of a TCR capable of binding to cell surface adhesion molecules, surface molecules of inflammatory cells seen in autoimmune diseases, or autoimmune-causing TCRs . As a non-limiting example, the targeting moiety of the present invention can be an scFv antibody that recognizes a tumor-specific antigen (TSA), such as an scFv antibody that specifically recognizes and binds to human mesothelin antibodies SS, SS1, and HN1, or GD2. scFv, CD19 antigen binding domain, NKG2D ligand binding domain, human anti-mesothelin scFv, anti-CS1 binder, anti-BCMA binding domain, anti-CD19 scFv antibody, GFRα4 antigen-binding fragment, anti-CLL-1 (type C agglutination 1) binding domain, CD33 binding domain, GPC3 (glypican-3) binding domain, GFRα4 (glycosyl-phosphatidylinositol (GPI)-linked GDNF family α-receptor 4 cell surface receptor) binding domain, CD123 binding domain, anti-ROR1 antibody or fragment thereof, scFv specific for GPC-3, scFv against CSPG4 and scFv against folate receptor α. [Intracellular signaling domain]

[0179] The intracellular domain of the CAR fusion polypeptide transmits a signal to immune effector cells after binding to its target molecule, activating at least one of the normal effector functions of immune effector cells, including lytic activity (e.g., interleukin secretion) or auxiliary active. Therefore, the intracellular domain includes the "intracellular signaling domain" of the T cell receptor (TCR).

[0180] In some aspects, an intact intracellular signaling domain may be employed. In other aspects, truncated portions of the intracellular signaling domain may be used in place of the intact chain, as long as they transduce effector function signals.

[0181] In some embodiments, the intracellular signaling domain may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). Examples of ITAM-containing cytoplasmic signaling sequences include those derived from TCR CD3 [ζ], FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b and CD66d. In one example, the intracellular signaling domain is the CD3 zeta (CD3 zeta) signaling domain.

[0182] In some embodiments, the intracellular domain further includes one or more costimulatory signaling domains that provide additional signals to immune effector cells. These costimulatory signaling domains, combined with signaling domains, can further improve the expansion, activation, memory, persistence and tumor eradication efficiency of CAR-engineered immune cells (such as CAR T cells). In some cases, the costimulatory signaling domain contains 1, 2, 3, or 4 cytoplasmic domains with one or more intracellular signaling and / or costimulatory molecules. The costimulatory signaling domain can be the intracellular / cytoplasmic domain of costimulatory molecules, including but not limited to CD2, CD7, CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, ICOS (CD278), GITR (glucocorticoid-induced tumor necrosis factor receptor), LFA-1 (lymphocyte function-associated antigen-1), LIGHT, NKG2C, B7-H3. In one example, the costimulatory signaling domain is derived from the cytoplasmic domain of CD28. In another example, the costimulatory signaling domain is derived from the cytoplasmic domain of 4-1BB (CD137). In another example, the costimulatory signaling domain can be the intracellular domain of GITR as taught in U.S. Patent No. 9,175,308; the contents of which are incorporated herein by reference in their entirety.

[0183] In some embodiments, the intracellular region may comprise a functional signaling domain from a protein selected from the group consisting of MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, interleukin receptors, integrins Factors, signaling lymphoid activated proteins (SLAM), such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, CD2F-10, SLAMF6, SLAMF7, activated NK cell receptor, BTLA, Toll ligand Receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2R β, IL2R γ, IL7R α, IL-15Ra, ITGA4 , VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18 , LFA-1, ITGB7, NKG2D, NKG2C, NKD2C SLP76, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 ( BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, CD270 (HVEM) , GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, DAP 10, TRIM, ZAP70, killer immunoglobulin receptors (KIR) such as KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3 and KIR2DP1; lectin-related NK cell receptors such as Ly49, Ly49A and Ly49C.

[0184] In some embodiments, the intracellular signaling domain of the invention may comprise a signaling domain derived from JAK-STAT. In other embodiments, the intracellular signaling domain of the invention may comprise a signaling domain derived from DAP-12 (death associated protein 12) (Topfer et al., Immunol., 2015, 194: 3201-3212; and Wang et al. Human, Cancer Immunol., 2015, 3: 815-826). DAP-12 is a key signal transduction receptor in NK cells. Activation signaling mediated by DAP-12 plays an important role in triggering NK cell cytotoxic responses against certain tumor cells and virus-infected cells. The cytoplasmic domain of DAP12 contains an immunoreceptor tyrosine-based activation motif (ITAM). Therefore, a CAR containing a DAP12-derived signaling domain can be used to transfer NK cells receptively. [Transmembrane domain]

[0185] In some embodiments, a CAR may comprise a transmembrane domain. As used herein, the term "transmembrane domain (TM)" refers broadly to an amino acid sequence of about 15 residues in length that spans the plasma membrane. The transmembrane domain may include at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid residues and spans the plasma membrane. In some embodiments, the transmembrane domain can be derived from natural or synthetic sources. The transmembrane domain of a CAR can be derived from any natural membrane-binding protein or transmembrane protein. For example, the transmembrane region may be derived from (ie, include at least the transmembrane region thereof): the alpha, beta or zeta chain of a T cell receptor, CD3ε, CD4, CD5, CD8, CD8α, CD9 , CD16, CD22, CD33, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152 or CD154.

[0186] Alternatively, the transmembrane domains of the invention can be synthetic. In some aspects, the synthetic sequence may contain primarily hydrophobic residues, such as leucine and valine.

[0187] In some embodiments, the transmembrane domain may be selected from the group consisting of: CD8α transmembrane domain, CD4 transmembrane domain, CD28 transmembrane domain, CTLA-4 transmembrane domain, PD-1 transmembrane domain, and human IgG4 Fc region .

[0188] In some embodiments, a CAR may include an optional hinge region (also known as a spacer). Hinge sequences are flexible short amino acid sequences that facilitate movement of the target binding domain away from the effector cell surface to enable appropriate cell / cell contact, target binding, and effector cell activation of the extracellular targeting domain. A hinge sequence can be placed between the targeting moiety and the transmembrane domain. The hinge sequence may be any suitable sequence derived or obtained from any suitable molecule. The hinge sequence can be derived from all or part of the hinge region of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4), that is, the sequence falls between the CH1 and CH2 domains of the immunoglobulin, such as IgG4 Fc hinge, type 1 membrane protein (such as CD8α CD4, CD28 and CD7), which can be a wild-type sequence or a derived sequence. Some hinge regions include immunoglobulin CH3 domains or both CH3 and CH2 domains. In certain embodiments, the hinge region may be modified relative to IgG1, IgG2, IgG3, or IgG4, including one or more substitutions with amino acid residues different from those present in the unmodified hinge. amino acid residues, such as 1, 2, 3, 4 or 5 residues.

[0189] In some embodiments, a CAR may contain one or more linkers between any domains of the CAR. The length of the linker can range from 1 to 30 amino acids. In this regard, the length of the linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. In other embodiments, the connectors may be flexible.

[0190] In some embodiments, components including a targeting moiety, a transmembrane domain, and an intracellular signaling domain can be constructed in a single fusion polypeptide. Fusion polypeptides can be the payload of the effector module of the invention.

[0191] In some embodiments, the carrier or payload may be or encode a CD19-specific CAR targeting different B-cell malignancies and a HER2-specific CAR targeting sarcoma, glioblastoma, and advanced Her2-positive lung malignancies. . Tandem CAR (TanCAR)

[0192] In some embodiments, the CAR can be a tandem chimeric antigen receptor (TanCAR) capable of targeting two, three, four, or more tumor-specific antigens. In some aspects, the CAR is a bispecific TanCAR that includes two targeting domains that recognize two different TSA's on tumor cells. Bispecific TanCAR can be further defined as comprising an extracellular domain containing a targeting domain specific for a first tumor antigen (e.g., an antigen recognition domain) and a targeting domain specific for a second tumor antigen (e.g., an antigen recognition domain). district. In other aspects, the CAR is a multispecific TanCAR including three or more targeting domains configured in a tandem arrangement. The length of the space between the targeting domains in TanCAR can be between about 5 and about 30 amino acids, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 amino acids. [Cracked] [CAR]

[0193] In some embodiments, a CAR component, including a targeting moiety, a transmembrane domain, and an intracellular signaling domain, can be split into two or more parts such that it relies on factors that promote the assembly of a fully functional receptor. Multiple inputs. As a non-limiting example, a split CAR consists of two parts assembled in a small molecule-dependent manner; one part of the receptor is characterized by an extracellular antigen-binding domain (e.g., scFv) and the other part has an intracellular signaling domain , such as the CD3ζ intracellular domain.

[0194] In other aspects, split portions of the CAR system can be further modified to increase signal. As a non-limiting example, the second portion of the cytoplasmic fragment can be anchored to the plasma membrane by incorporating a transmembrane domain (eg, a CD8 alpha transmembrane domain) into the construct. Additional extracellular domains can also be added to the second part of the CAR system, such as those that mediate homodimerization. These modifications can increase receptor output activity, that is, T cell activation.

[0195] In some embodiments, the two parts of the split CAR system contain heterodimerization domains that conditionally interact upon binding of the heterodimeric small molecule. Thus, receptor components assemble in the presence of small molecules to form a complete system that can then be activated by antigen conjugation. Any known heterodimeric component can be incorporated into the split CAR system. Other small molecule-dependent heterodimerization domains can also be used, including but not limited to gibberellin-induced dimerization system (GID1-GAI), trimethoprim-SLF-induced ecDHFR and FKBP dimerization. and ABA (abscisic acid)-induced dimerization of PP2C and PYL domains. Dual regulation of induced assembly (e.g., ligand-dependent dimerization) and degradation (e.g., domain destabilization-induced CAR degradation) using split CAR systems may provide more flexibility to control CAR-modified T cell activity. [switchable] [CAR]

[0196] In some embodiments, a CAR can be a switchable CAR that can be temporarily switched on to a controllable CAR in response to a stimulus (eg, a small molecule). In this CAR design, the system is integrated directly into the hinge domain that separates the scFv domain from the cell membrane domain in the CAR. Such systems have the potential to split or combine different key functions of the CAR, such as activation and costimulation within different chains of the receptor complex, thus simulating the complexity of the natural TCR architecture. This integrated system switches scFv and antigen interactions between on / off states controlled by the absence / presence of stimuli. [reversible] [CAR]

[0197] In some embodiments, the CAR can be a reversible CAR system. In this CAR architecture, the LID (ligand-induced degradation) domain is incorporated into the CAR system. CAR can be temporarily down-regulated by adding ligands to the LID domain. [inhibitory] [CAR (iCAR)]

[0198] In some embodiments, the CAR can be an inhibitory CAR. Inhibitory CAR (iCAR) refers to a bispecific CAR design in which negative signaling is used to enhance tumor specificity and limit normal tissue toxicity. This design incorporates a second CAR with a surface antigen recognition domain and an inhibitory signaling domain to limit T cell reactivity even when activating receptors are simultaneously engaged. This antigen recognition domain targets normal tissue-specific antigens so that T cells can be activated in the presence of the first target protein, but if there is a second protein that binds to iCAR, T cell activation is inhibited.

[0199] As a non-limiting example, iCARs against prostate-specific membrane antigen (PMSA) based on CTLA4 and PD1 inhibitory domains exhibit the ability to selectively limit interleukin secretion, cytotoxicity and proliferation induced by T cell activation. [Chimeric switch receptor]

[0200] In some embodiments, the carrier or payload may be or encode a chimeric switch receptor that can switch a negative signal to a positive signal. As used herein, the term "chimeric switch receptor" refers to a fusion protein comprising a first extracellular domain and a second transmembrane and intracellular domain, wherein the first domain includes a negative signaling region and the second domain includes a positive intracellular signal conductive zone. In some aspects, the fusion protein is a chimeric switch receptor containing the extracellular domain of an inhibitory receptor on a T cell fused to the transmembrane and cytoplasmic domains of a costimulatory receptor. This chimeric switch receptor converts T cell inhibitory signals into T cell stimulatory signals.

[0201] As a non-limiting example, a chimeric switch receptor may comprise the extracellular domain of PD-1 fused to the transmembrane and cytoplasmic domains of CD28. In some aspects, the extracellular domains of other inhibitory receptors such as CTLA-4, LAG-3, TIM-3, KIR, and BTLA can also be combined with those derived from costimulatory receptors such as CD28, 4-1BB, CD27, OX40 , CD40, GTIR and ICOS) transmembrane and cytoplasmic domain fusion.

[0202] In some embodiments, chimeric switch receptors may include recombinant receptors containing: and stimulatory interleukin receptors such as IL-2R (IL-2Rα, IL-2Rβ, and IL-2Rγ) and IL-7Rα ) is fused to the extracellular interleukin-binding domain of inhibitory interleukin receptors (such as IL-13 receptor alpha (IL-13Rα1), IL-10R, and IL-4Rα) with the intracellular signaling domain. One example of such a chimeric interleukin receptor is a recombinant receptor containing the interleukin-binding extracellular domain of IL-4Ralpha linked to the intracellular signaling domain of IL-7Ralpha.

[0203] In some embodiments, the chimeric switch receptor can be a chimeric TGFβ receptor. A chimeric TGFβ receptor may comprise: an extracellular domain derived from a TGFβ receptor, such as TGFβ receptor 1, TGFβ receptor 2, TGFβ receptor 3, or any other TGFβ receptor or variant thereof; and a non-TGFβ receptor intracellular domain. area. The non-TGFβ receptor intracellular domain may be derived from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CD28, 4-1BB (CD137), OX40 (CD134), CD3ζ, CD40, CD27 or intracellular domains of combinations thereof or fragments thereof. [Activation conditionality] [CAR]

[0204] In some embodiments, the carrier or payload may be or encode an activated conditional chimeric antigen receptor that is expressed only in activated immune cells. The expression of a CAR can be coupled to an activating conditional control region that refers to one or more nucleic acid sequences that induce the transcription and / or expression of a sequence (eg, CAR) under its control. Such activation conditional control regions may be the promoters of genes that are upregulated during activation of immune effector cells, such as the IL2 promoter or NFAT binding site. [Targeting tumor cells with specific proteoglycan markers] [CAR]

[0205] In some embodiments, the carrier or payload may be or encode a CAR that targets a specific type of cancer cell. Human cancer cells and cancer metastases may express unique and otherwise abnormal proteoglycans, such as polysaccharide chains (e.g., chondroitin sulfate (CS), dermatan sulfate (DS or CSB), acetyl heparin sulfate (heparan) sulfate;HS) and heparin). Therefore, a CAR can be fused to a binding moiety that recognizes cancer-associated proteoglycans. In one example, a CAR can be fused to a VAR2 CSA polypeptide that binds with high affinity to a specific type of chondroitin A sulfate (CSA) linked to proteoglycans (VAR2-CAR). The extracellular ScFv portion of the CAR can be replaced by a VAR2CSA variant that contains at least the minimal CSA binding domain, thereby generating a CAR specific for chondroitin sulfate A (CSA) modification. Alternatively, the CAR can be fused to a split protein binding system to create a spy-CAR, in which the scFv portion of the CAR is replaced by a portion of the split protein binding system (such as SpyTag and Spy Trap), and the cancer recognition molecule (e.g., scFv and / or VAR2-CSA) is linked to the CAR via a split protein binding system. nucleic acid

[0206] The initial construct and the reference construct of the present invention may include a payload region (which may also be referred to as a carrier region), which is nucleic acid. The term "nucleic acid" in its broadest sense includes any compound and / or substance comprising a polymer of nucleotides that may be referred to as a polynucleotide. Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA) ) or its hybrid.

[0207] In some embodiments, the payload region includes nucleic acid sequences encoding more than one payload or payload.

[0208] In some embodiments, the payload region may be or encode a coding nucleic acid sequence.

[0209] In some embodiments, the payload region may be or encode a non-coding nucleic acid sequence.

[0210] In some embodiments, the payload region may be or encode both coding and non-coding nucleic acid sequences. [DNA, , ]

[0211] Deoxyribonucleic acid (DNA) is a molecule that carries the genetic information of all living things and is composed of two strands that wrap around each other to form a shape called a double helix. Each strand has a backbone composed of alternating sugar (deoxyribose) and phosphate groups. One of four bases is attached to each sugar: adenine (A), cytosine (C), guanine (G), and thymine (T). The two strands are held together by bonds between adenine and thymine or cytosine and guanine. The sequence of bases along the backbone serves as instructions for assembling proteins and RNA molecules.

[0212] In some embodiments, the payload region may be or encode coding DNA.

[0213] In some embodiments, the payload region may be or encode non-coding DNA.

[0214] In some embodiments, the payload region may be or encode both coding and non-coding DNA.

[0215] In some embodiments, DNA can be modified. Types of modifications include, but are not limited to, methylation, acetylation, phosphorylation, ubiquitination, and sumoylation. carrier

[0216] In some embodiments, the initial construct and / or the reference construct described herein may be or encoded by a vector (such as a plasmid or viral vector). In some embodiments, the initial construct and / or the reference construct is or encoded by a viral vector. Viral vectors may be, but are not limited to, herpes virus (HSV) vectors, retroviral vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, lentiviral vectors, and the like. In some embodiments, the viral vector is an AAV vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is an adenoviral vector. [adeno-associated virus] [(AAV)] [carrier]

[0217] Viruses of the Parvoviridae family are small, non-enveloped, icosahedral capsid viruses characterized by a single-stranded DNA genome. Parvoviridae viruses are composed of two subfamilies: Parvovirinae, which infects vertebrates, and Densovirinae, which infects invertebrates. Because their structure is relatively simple and can be easily manipulated using standard molecular biology techniques, this family of viruses is suitable as a biological tool. Viral genomes can be modified to contain minimal components for the assembly of functional recombinant viruses or virions that carry a desired payload or engineered to express or deliver a desired payload, which can be Delivery to target cells, tissues, organs or organisms.

[0218] The family Parvoviridae includes the genus Dependovirus, which includes adeno-associated viruses (AAV) that are capable of replicating in vertebrate hosts, including but not limited to humans, primates, cattle, Dog, horse and sheep species.

[0219] The AAV vector genome is a linear, single-stranded DNA (ssDNA) molecule approximately 5,000 nucleotides (nt) in length. The AAV vector genome may comprise a payload region and at least one inverted terminal repeat (ITR) or ITR region. ITRs are traditionally flanked by nucleotide sequences encoding nonstructural proteins (encoded by Rep genes) and structural proteins (encoded by capsid or Cap genes). While not wishing to be bound by theory, AAV vector genomes typically contain two ITR sequences. The AAV vector genome contains a characteristic T-shaped hairpin structure, which is defined by the self-complementary end 145 nucleotides of the 5' and 3' ends of ssDNA, forming an energetically stable double-stranded region. The double-stranded hairpin structure contains multiple functions, including but not limited to serving as an origin of DNA replication by serving as a primer for the endogenous DNA polymerase complex of the host virus replicating cell.

[0220] In addition to the encoded heterologous payload, the AAV vector genome may contain all or part of any naturally occurring and / or recombinant AAV serotype nucleotide sequence or variant. AAV variants may have significant sequence homology at the nucleic acid level (genome or capsid) and the amino acid level (capsid) to produce substantially physically and functionally equivalent constructs that are constructed by analogy Mechanisms are copied and assembled by similar mechanisms. Chiorini et al., J. Vir. 71: 6823-33 (1997); Srivastava et al., J. Vir. 45:555-64 (1983); Chiorini et al., J. Vir. 73:1309-1319 (1999) ; Rutledge et al., J. Vir. 72:309-319 (1998); and Wu et al., J. Vir. 74: 8635-47 (2000). The contents of each of these documents are cited in full. method is incorporated into this article.

[0221] In some embodiments, the AAV vector genome includes at least one control element that provides for replication, transcription, and translation of the coding sequence encoded therein. Not all control elements need to be present at all times, as long as the coding sequence is capable of replication, transcription and / or translation in a suitable host cell. Non-limiting examples of expression control elements include sequences for transcription initiation and / or termination, promoter and / or enhancer sequences, efficient RNA processing signals (such as splicing and polyadenylation signals), stabilization of cytoplasmic mRNA Sequences, sequences that enhance translational efficacy (e.g., Kozak consensus sequences), sequences that enhance protein stability, and / or sequences that enhance protein processing and / or secretion.

[0222] The AAV vector genome of the present invention can be produced in a recombinant manner and can be based on adeno-associated virus (AAV) parent or reference sequences. As used herein, a "vector genome" is any molecule or portion that transports, transduces, or otherwise serves as a vector for a heterologous molecule, such as a nucleic acid described herein.

[0223] In addition to single-stranded AAV vector genomes (eg, ssAAV), the present invention also provides self-complementary AAV (scAAV) vector genomes. The scAAV vector genome contains DNA strands that are annealed together to form double-stranded DNA. By skipping second-strand synthesis, scAAV achieves rapid expression in cells.

[0224] In some embodiments, the AAV vector genome is scAAV.

[0225] In some embodiments, the AAV vector genome is ssAAV.

[0226] In some embodiments, the AAV vector genome can be part of an AAV particle, in which the serotype of the capsid can be, but is not limited to, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43- 1. AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7 / rh.48, AAV1-8 / rh.49, AAV2-15 / rh.62, AAV2-3 / rh.61, AAV2-4 / rh. 50. AAV2-5 / rh.51, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3-11 / rh.53, AAV4-8 / r11.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.10, AAV16. 12 / hu.11, AAV29.3 / bb.1, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.40, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.10 / hu. 60. AAV161.6 / hu.61, AAV33.12 / hu.17, AAV33.4 / hu.15, AAV33.8 / hu.16, AAV52 / hu.19, AAV52.1 / hu.20, AAV58. 2 / hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2, AAVC5, AAV-DJ, AAV-DJ8, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi.1, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh. 59. AAVhu.12, AAVH6, AAVLK03, AAVH-1 / hu.1, AAVH-5 / hu.3, AAVLG-10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy. 6. AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu. 29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48R1, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu. 54. AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14 / 9, AAVhu. t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19 , AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh .37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh .52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74 , AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, sheep AAV, bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35 , AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T, AAV-PAEC, AAV -LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13 , AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV -PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100 -7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10 , BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu. 28. AAV46.6 / hu.29, AAV128.1 / hu.43, true AAV (ttAAV), UPENN AAV 10, Japanese AAV 10 serotype, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8. AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd- 3. AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd- B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-H1, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd- N4, AAV CKd-N9, AAV CLg-F1, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv- 1. AAV CLv1-1, AAV Clv1-10, AAV CLv1-2, AAV CLv-12, AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV Clv1-7, AAV Clv1-8, AAV Clv1- 9. AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-D1, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv- D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-E1, AAV CLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv- L6, AAV CLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-R1, AAV CLv- R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp- 11. AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp- 8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7 , AAVF8 / HSC8, AAVF9 / HSC9, PHP.B, PHP.A, G2B-26, G2B-13, TH1.1-32 and / or TH1.1-35, and their variants. .

[0227] Inverted terminal repeat (ITR)

[0228] In some embodiments, the AAV vector genome can include at least one ITR region and a payload region. In some embodiments, the vector genome has two ITRs. The two ITRs flank the payload area at the 5' and 3' ends. The ITR serves as the origin of replication and contains the recognition site for replication. ITRs contain complementary and symmetrically configured sequence regions. The ITR incorporated into the vector genome of the invention may comprise a naturally occurring polynucleotide sequence or a recombinantly derived polynucleotide sequence.

[0229] The ITR can be derived from the same serotype as the capsid or a derivative thereof. The ITR can be of a different serotype than the capsid. In some embodiments, AAV particles have more than one ITR. In a non-limiting example, the AAV particle has a vector genome containing two ITRs. In some embodiments, the ITRs are of the same serotype as each other. In another embodiment, the ITRs are of different serotypes. Non-limiting examples include zero, one or two of the ITRs being of the same serotype as the capsid. In some embodiments, both ITRs of the vector genome of the AAV particle are AAV2 ITRs.

[0230] Independently, each ITR can be about 100 to about 150 nucleotides in length. The ITR can be about 100-105 nucleotides in length, 106-110 nucleotides in length, 111-115 nucleotides in length, 116-120 nucleotides in length, 121-125 nucleotides in length Nucleotides, 126-130 nucleotides in length, 131-135 nucleotides in length, 136-140 nucleotides in length, 141-145 nucleotides in length or 146-150 nucleotides in length Nucleotides. In some embodiments, the ITR is 140 to 142 nucleotides in length. Non-limiting examples of ITR lengths are 102, 140, 141, 142, 145 nucleotides in length, and those having at least 95% identity thereto. [Promoter]

[0231] In some embodiments, the payload region of the vector genome includes at least one element that enhances transgene target specificity and expression (see, e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015 ;The contents of which are incorporated herein by reference in their entirety). Non-limiting examples of elements that enhance target specificity and expression of transgenic genes include promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (PolyA) signal sequences and upstream enhancers (USE), CMV enhancer and intron.

[0232] In some embodiments, a promoter is considered effective when it drives the expression of a polypeptide encoded in the payload region of the vector genome of an AAV particle.

[0233] In certain embodiments, a promoter is considered to be a valid promoter when it drives expression in the cell to which it is targeted.

[0234] In some embodiments, the promoter drives expression of the payload in the targeted tissue for a period of time. Promoter-driven performance can be maintained for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days , 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months Month, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or a period exceeding 10 years. Performance lasts 1 to 5 hours, 1 to 12 hours, 1 to 2 days, 1 to 5 days, 1 to 2 weeks, 1 to 3 weeks, 1 to 4 weeks, 1 to 2 months, 1 to 4 months, 1 to 6 months, 2 to 6 months, 3 to 6 months, 3 to 9 months, 4 to 8 months, 6 to 12 months, 1 to 2 years, 1 to 5 years, 2 to 5 years , 3 to 6 years, 3 to 8 years, 4 to 8 years or 5 to 10 years.

[0235] In some embodiments, the promoter-driven payload performs for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months , 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years , 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 Years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 years, 49 years, 50 years, 55 years, 60 years, 65 years or more than 65 years.

[0236] Promoters may be naturally occurring or non-naturally occurring. Non-limiting examples of promoters include viral promoters, plant promoters, and mammalian promoters. In some embodiments, the promoter can be a human promoter. In some embodiments, the promoter can be truncated.

[0237] Promoters that drive or promote expression in most tissues include, but are not limited to, human elongation factor 1 alpha-subunit (EF1 alpha), cytomegalovirus (CMV) immediate early enhancer and / or promoter, chicken beta-actin ( CBA) and its derivatives CAG, β-glucuronidase (GUSB) or ubiquitin C (UBC). Tissue-specific expression elements can be used to limit expression to certain cell types, such as, but not limited to, muscle-specific promoters, B cell promoters, monocyte promoters, leukocyte promoters, macrophage promoters, pancreatic gland Alveolar cell promoter, endothelial cell promoter, lung tissue promoter, astrocyte promoter, or nervous system promoter that can be used to limit expression of neurons, astrocytes, or oligodendritic glial cells .

[0238] Non-limiting examples of muscle-specific promoters include mammalian muscle creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, mammalian troponin I (TNNI2) promoter, and mammalian bone Alpha-actin (ASKA) promoter (see, eg, U.S. Patent Publication US20110212529, the contents of which are incorporated herein by reference in their entirety).

[0239] Non-limiting examples of tissue-specific expression elements of neurons include neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), synaptophysin (Syn ), methyl-CpG binding protein 2 (MeCP2), Ca 2+ / calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurociliary light chain (NFL) or heavy chain (NFH), β-hemoglobulin small gene nβ2, proproenkephalin (PPE), enkephalin (Enk) and agonistic amino acid transporter 2 (EAAT2) promoters. Non-limiting examples of tissue-specific expression elements of astrocytes include glial fibrillary acidic protein (GFAP) and EAAT2 promoter. Non-limiting examples of tissue-specific expression elements of oligodendritic cells include the myelin basic protein (MBP) promoter.

[0240] In some embodiments, the promoter may be less than 1 kb. The length of the promoter can be 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more than 800 nucleotides. The length of the promoter can be 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, Between 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800 or 700-800.

[0241] In some embodiments, the promoter may be a combination of two or more components of the same or different origin or parental promoters such as, but not limited to, CMV and CBA. Each component may have 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 381, 382, ​​383, 384 ,385,386,387,388,389,390,400,410,420,430,440,450,460,470,480,490,500,510,520,530,540,550,560,570,580 , 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or a length exceeding 800. The length of each component can be between 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300- Between 800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800 or 700-800. In some embodiments, the promoter is a combination of a 382-nucleotide CMV-enhancer sequence and a 260-nucleotide CBA-promoter sequence.

[0242] In some embodiments, the vector genome contains a ubiquitous promoter. Non-limiting examples of ubiquitous promoters include CMV, CBA (including derivatives CAG, CBh, etc.), EF-1α, PGK, UBC, GUSB (hGBp), and UCOE (promoter of HNRPA2B1-CBX3).

[0243] In some embodiments, the promoter is not cell specific.

[0244] In some embodiments, the vector genome includes an engineered promoter.

[0245] In some embodiments, the vector genome contains a promoter from a naturally occurring protein. [Untranslated area] [(] [UTR] [)]

[0246] By definition, the wild-type untranslated region (UTR) of a gene is transcribed but not translated. Generally speaking, the 5' UTR starts at the transcription start site and ends at the initiation codon, and the 3' UTR starts immediately after the stop codon and continues until the transcription termination signal.

[0247] Features typically found in well-expressed genes of a specific target organ can be engineered into the UTR to enhance stability and protein production. As non-limiting examples, from mRNAs commonly expressed in the liver (e.g., albumin, serum amyloid A, lipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII) The 5' UTR can be used in the vector genome of the AAV particles of the present invention to enhance the performance in liver cell lines or liver.

[0248] While not wishing to be bound by theory, the wild-type 5' untranslated region (UTR) includes features that function in the initiation of translation. Kozak sequences are often included in the 5' UTR, and Kozak sequences are generally known to be involved in the process by which ribosomes initiate translation of multiple genes. The Kozak sequence has the common CCR(A / G)CCAUGG, where R is the purine (adenine or guanine) three bases upstream of the start codon (ATG), followed by another "G".

[0249] In some embodiments, the 5' UTR in the vector genome includes a Kozak sequence.

[0250] In some embodiments, the 5'UTR in the vector genome does not include the Kozak sequence.

[0251] While not wishing to be bound by theory, it is known that the wild-type 3' UTR has fragments of adenosine and uridine embedded in it. Such AU-rich tags are particularly prevalent in genes with high turnover rates. AU-rich elements (AREs) can be divided into three categories based on their sequence characteristics and functional properties (Chen et al., 1995, the contents of which are incorporated herein by reference in full): Class I AREs, such as but not limited to c-Myc and MyoD , there are several scattered copies of AUUUA primitives in the U-rich region. Class II AREs, such as but not limited to GM-CSF and TNF-a, have two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Class III ARES, such as but not limited to c-Jun and myogenin, are less well defined. These U-rich regions do not contain the AUUUA motif. Most proteins that bind to the ARE are known to destabilize the message, while members of the ELAV family (most notably, HuR) have been documented to increase mRNA stability. HuR binds to all three classes of AREs. Engineering HuR-specific binding sites into the 3' UTR of nucleic acid molecules will cause HuR binding, thereby causing message stabilization in vivo.

[0252] The introduction, removal, or modification of 3' UTR AU-rich elements (AREs) can be used to modulate the stability of polynucleotides. When engineering a specific polynucleotide, such as the payload region of a vector genome, one or more copies of the ARE can be introduced to reduce polynucleotide stability and thereby reduce translation of the resulting protein and reduce its output. Likewise, AREs can be identified and removed or mutated to increase intracellular stability, and thus increase translation and production of the resulting protein.

[0253] In some embodiments, the 3' UTR of the vector genome may include an oligo(dT) sequence for templated addition of a polyA tail.

[0254] In some embodiments, the vector genome may include at least one miRNA seed, binding site, or complete sequence. MicroRNA (or miRNA or miR) is a 19-25 nucleotide non-coding RNA that binds to the site of a nucleic acid target and downregulates gene expression by reducing the stability of the nucleic acid molecule or by inhibiting translation. The microRNA sequence contains the "seed" region, that is, the sequence in the region of positions 2-8 of the mature microRNA, which has perfect Watson-Crick complementarity with the nucleic acid's miRNA target sequence. .

[0255] In some embodiments, the vector genome can be engineered to include, alter, or remove at least one miRNA binding site, sequence, or seed region.

[0256] Any UTR from any gene known in the art can be incorporated into the vector genome of the AAV particle. These UTRs, or portions thereof, may be placed in the same orientation as in the gene from which they are selected, or their orientation or position may vary. In some embodiments, the UTR used in the vector genome of the AAV particle may be inverted, shortened, elongated, or made with one or more other 5' UTRs or 3' UTRs known in the art. As used herein, the term "altered" when related to a UTR means that the UTR has changed in some way relative to the reference sequence. For example, the 3' or 5' UTR may be altered relative to the wild-type or native UTR based on changes in orientation or position as taught above, or may be modified by including additional nucleotides, nucleotide deletions, nucleotide deletions, Changed by exchange or transposition.

[0257] In some embodiments, the vector genome of the AAV particle includes at least one artificial UTR that is not a variant of the wild-type UTR.

[0258] In some embodiments, the vector genome of the AAV particle includes UTRs selected from a family of transcripts in which proteins share common functions, structures, features or properties. [polyadenylation sequence]

[0259] In some embodiments, the vector genome includes at least one polyadenylation sequence between the 3' end of the payload coding sequence and the 5' end of the 3' ITR.

[0260] In some embodiments, the polyadenylation (polyA) sequence can range from absent to about 500 nucleotides in length. The polyadenylation sequence length may be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45 ,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70 ,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95 ,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120 ,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145 ,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170 ,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195 ,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220 ,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245 ,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270 ,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291,292,293,294,295 ,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317,318,319,320 ,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342,343,344,345 ,346,347,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370 ,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,391,392,393,394,395 ,396,397,398,399,400,401,402,403,404,405,406,407,408,409,410,411,412,413,414,415,416,417,418,419,420 ,421,422,423,424,425,426,427,428,429,430,431,432,433,434,435,436,437,438,439,440,441,442,443,444,445 ,446,447,448,449,450,451,452,453,454,455,456,457,458,459,460,461,462,463,464,465,466,467,468,469,470 ,471,472,473,474,475,476,477,478,479,480,481,482,483,484,485,486,487,488,489,490,491,492,493,494,495 , 496, 497, 498, 499 and 500 nucleotides.

[0261] In some embodiments, the polyadenylation sequence is 50-100 nucleotides in length. In some embodiments, the polyadenylation sequence is 50-150 nucleotides in length. In some embodiments, the polyadenylation sequence is 50-160 nucleotides in length. In some embodiments, the polyadenylation sequence is 50-200 nucleotides in length. In some embodiments, the polyadenylation sequence is 60-100 nucleotides in length. In some embodiments, the polyadenylation sequence is 60-150 nucleotides in length. In some embodiments, the polyadenylation sequence is 60-160 nucleotides in length. In some embodiments, the polyadenylation sequence is 60-200 nucleotides in length. In some embodiments, the polyadenylation sequence is 70-100 nucleotides in length. In some embodiments, the polyadenylation sequence is 70-150 nucleotides in length. In some embodiments, the polyadenylation sequence is 70-160 nucleotides in length. In some embodiments, the polyadenylation sequence is 70-200 nucleotides in length. In some embodiments, the polyadenylation sequence is 80-100 nucleotides in length. In some embodiments, the polyadenylation sequence is 80-150 nucleotides in length. In some embodiments, the polyadenylation sequence is 80-160 nucleotides in length. In some embodiments, the polyadenylation sequence is 80-200 nucleotides in length. In some embodiments, the polyadenylation sequence is 90-100 nucleotides in length. In some embodiments, the polyadenylation sequence is 90-150 nucleotides in length. In some embodiments, the polyadenylation sequence is 90-160 nucleotides in length. In some embodiments, the polyadenylation sequence is 90-200 nucleotides in length. [connector]

[0262] The vector genome can be engineered with one or more spacer or linker regions to separate coding or non-coding regions.

[0263] In some embodiments, the payload region of the vector genome optionally encodes one or more linker sequences. In some cases, the linker can be a peptide linker that can be used to join the polypeptide encoded by the payload region (i.e., during expression, the antibody light and heavy chains). Some peptide linkers can be cleaved into separate heavy and light chain domains after expression, allowing assembly of mature antibodies or antibody fragments. Linker cleavage can be enzymatic. In some cases, the linker contains enzymatic cleavage sites to facilitate intracellular or extracellular cleavage. Some payload regions encode linkers that interrupt polypeptide synthesis during translation of the linker sequence from the mRNA transcript. Such linkers facilitate translation of individual protein domains from a single transcript. In some cases, two or more linkers are encoded by the payload region of the vector genome.

[0264] An internal ribosome entry site (IRES) is a nucleotide sequence (>500 nucleotides) that allows initiation of translation in the middle of the mRNA sequence (Kim, J.H. et al., 2011. PLoS One 6(4): e18556; The contents are incorporated herein by reference in their entirety). The use of an IRES sequence ensures co-expression of genes preceding and following the IRES, although the sequence following the IRES may be transcribed and translated at a lower level than the sequence preceding the IRES.

[0265] 2A peptides are small "self-cleaving" peptides (18-22 amino acids) derived from viruses such as foot-and-mouth disease virus (F2A), porcine Jeshin virus-1 (P2A), Tetrahydropterus virus (T2A) or horse Rhinitis virus type A (E2A) virus. The 2A designation specifically refers to the region of the picornavirus polymeric protein that causes ribosome skipping at the glycolyl-prolyl bond in the C-terminus of the 2A peptide (Kim, J.H. et al., 2011. PLoS One 6(4) ): e18556; the contents of which are incorporated herein by reference in full). This jump causes cleavage between the 2A peptide and its immediately downstream peptide. In contrast to the IRES linker, the 2A peptide results in stoichiometric expression of the protein flanked by the 2A peptide, and its shorter length can be advantageous in generating viral expression vectors.

[0266] Some payload regions encode linkers containing furin cleavage sites. Furin is a calcium-dependent endoserine protease that cleaves proteins just downstream of the basic amino acid target sequence (Arg-X-(Arg / Lys)-Arg) (Thomas, G., 2002. Nature Reviews Molecular Cell Biology 3(10): 753-66; the contents of which are incorporated herein by reference in full). Furin is enriched in the trans-Golgi network, where it is involved in processing cellular precursor proteins. Furin also plays a role in activating a variety of pathogens. This activity can be exploited to express the polypeptides of the invention.

[0267] In some embodiments, the payload region may encode one or more linkers that include cathepsin, matrix metalloproteinase, or legumin cleavage sites. Such linkers are described, for example, by Cizeau and Macdonald in International Publication No. WO2008052322, the contents of which are incorporated herein by reference in their entirety. Cathepsins are a family of proteases with a unique mechanism for cleaving specific proteins. Cathepsin B is a cysteine ​​protease, and cathepsin D is an asparaginyl protease. Matrix metalloproteinases are a family of calcium-dependent and zinc-containing endopeptidases. Legumin is an enzyme that catalyzes the hydrolysis of (-Asn-Xaa-) bonds between proteins and small molecule substrates.

[0268] In some embodiments, the payload region may encode an uncleaved linker. Such linkers may include simple amino acid sequences, such as glycine-rich sequences. In some cases, the linker may comprise a flexible peptide linker comprising glycine and serine residues. The linker can include flexible peptide linkers of varying lengths, such as nxG4S, where n=1-10, and the length of the encoded linker varies between 5 and 50 amino acids. In a non-limiting example, the linker can be 5xG4S. These flexible linkers are small and have no side chains, so they tend not to affect secondary protein structure while providing a flexible linker between antibody segments (George, R.A. et al., 2002. Protein Engineering 15(11): 871-9; Huston, J.S. et al., 1988. PNAS 85:5879-83; and Shan, D. et al., 1999. Journal of Immunology. 162(11):6589-95; Its contents Each is incorporated herein by reference in its entirety). In addition, the polarity of the serine residues improves solubility and prevents aggregation problems.

[0269] In some embodiments, the payload region of the invention may encode a small and non-branched serine-rich peptide linker, such as that described by Huston et al. in U.S. Patent No. 5,525,491, the contents of which are incorporated by reference in their entirety. incorporated herein. Polypeptides encoded by the payload region of the invention and linked by serine-rich linkers have increased solubility.

[0270] In some embodiments, the payload region of the present invention may encode artificial linkers, such as those described by Whitlow and Filpula in US Patent No. 5,856,456 and by Ladner et al. in US Patent No. 4,946,778. Otherwise, their respective contents are incorporated into this article by full reference. [Intron]

[0271] In some embodiments, the payload region contains at least one element for enhancing performance, such as one or more introns or portions thereof. Non-limiting examples of introns include MVM (67-97 bp), F. ), adenovirus splice donor / immunoglobulin splice acceptor (500 bp), SV40 late splice donor / splice acceptor (19S / 16S) (180 bp) and hybrid adenovirus splice donor / IgG splice acceptor ( 230 bp).

[0272] In some embodiments, an intron or intron portion may be 100-500 nucleotides in length. The length of the intron can be 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 200 ,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,450 , 460, 470, 480, 490 or 500. The length of the intron can range from 80-100, 80-120, 80-140, 80-160, 80-180, 80-200, 80-250, 80-300, 80-350, 80-400, 80- Between 450, 80-500, 200-300, 200-400, 200-500, 300-400, 300-500 or 400-500. [Lentiviral vector]

[0273] Lentiviral vectors are a type of retrovirus that can infect both dividing and non-dividing cells because their viral capsid can penetrate the intact membrane of the target cell's nucleus. Lentiviral vectors have the ability to deliver transgenic genes into long-lasting tissues that cannot be treated with stable gene manipulation. Lentiviral vectors have also opened up new perspectives for gene therapy of a wide range of hereditary and acquired conditions, and real proposals for their clinical use appear to be imminent. RNA

[0274] Ribonucleic acid (RNA) is a molecule composed of nucleotides in the form of ribose linked to nitrogenous bases and phosphate groups. Nitrogenous bases include adenine (A), guanine (G), uracil (U) and cytosine (C). Generally speaking, RNA mainly exists in single-stranded form, but in some cases it can also exist in double-stranded form. The length, form, and structure of RNA vary depending on the purpose of the RNA. For example, RNA can vary in length from short sequences (e.g., siRNA) to long sequences (e.g., lncRNA), can be linear (e.g., mRNA) or circular (e.g., oRNA), and can be coding (e.g., mRNA) or non-coding (e.g. lncRNA) sequence.

[0275] In some embodiments, the payload region may be or encode coding RNA.

[0276] In some embodiments, the payload region may be or encode non-coding RNA.

[0277] In some embodiments, the payload region may be or encode both coding and non-coding RNA.

[0278] In some embodiments, the payload region includes nucleic acid sequences encoding more than one payload or payload.

[0279] In some embodiments, the payload region includes nucleic acid sequences that enhance expression of the gene. As a non-limiting example, the nucleic acid sequence is messenger RNA (mRNA). As another non-limiting example, the nucleic acid sequence is circular RNA (oRNA).

[0280] In some embodiments, the payload region includes a nucleic acid sequence that reduces or inhibits the expression of a gene. As non-limiting examples, the nucleic acid sequence is small interfering RNA (siRNA) or microRNA (miRNA). [messenger] [RNA (mRNA)]

[0281] In some embodiments, the initial construct and / or the baseline construct may be mRNA. As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a target of interest and is capable of being translated to produce the encoded target of interest in vitro, in vivo, in situ, or ex vivo.

[0282] Generally speaking, an mRNA molecule includes at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly-A tail. In some aspects, the RNA may include one or more structural and / or chemical modifications or changes that may reduce the innate immune response of the cell into which the mRNA is introduced. As used herein, a "structural" modification is a modification in which two or more linked nucleotides are inserted, deleted, duplicated, inverted, or randomized in a nucleic acid without significant chemical modification of the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect the structural modification, the structural modification is of a chemical nature and is therefore a chemical modification. However, structural modifications will produce different nucleotide sequences. For example, the polynucleotide "ATCG" can be chemically modified to "AT-5meC-G".

[0283] Generally speaking, the minimum length of the region of the initial construct and / or the reference construct may be a nucleic acid sufficient to encode a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. The length of the sequence. In another embodiment, the length may be sufficient to encode a peptide of 2-30 amino acids, such as 5-30, 10-30, 2-25, 5-25, 10-25 or 10-20 amino acids. . The length may be sufficient to encode a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25, or 30 amino acids, or no longer than 40 amino acids, such as no longer than 35, 30, 25, 20, Peptides of 17, 15, 14, 13, 12, 11 or 10 amino acids.

[0284] Generally, the region of the mRNA encoding the target of interest is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120 , 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,00 0 , 2,500 and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,0 00 nucleotides).

[0285] In some embodiments, the mRNA includes about 30 to about 100,000 nucleotides (e.g., 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 1,000, 30 to 1,500, 30 to 3,000, 30 to 5,000 , 30 to 7,000, 30 to 10,000, 30 to 25,000, 30 to 50,000, 30 to 70,000, 100 to 250, 100 to 500, 100 to 1,000, 100 to 1,500, 100 to 3,000, 100 to 5,000, 100 to 7,000, 100 to 10,000, 100 to 25,000, 100 to 50,000, 100 to 70,000, 100 to 100,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 500 to 5,000, 500 to 7,000, 500 to 10,00 0. 500 to 25,000 , 500 to 50,000, 500 to 70,000, 500 to 100,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 5,000, 1,000 to 7,000, 1,000 to 10,000, 1,000 to 25,000 , 1,000 to 50,000, 1,000 to 70,000 , 1,000 to 100,000, 1,500 to 3,000, 1,500 to 5,000, 1,500 to 7,000, 1,500 to 10,000, 1,500 to 25,000, 1,500 to 50,000, 1,500 to 70,000, 1,500 to 100,000, 2,0 00 to 3,000, 2,000 to 5,000, 2,000 to 7,000 , 2,000 to 10,000, 2,000 to 25,000, 2,000 to 50,000, 2,000 to 70,000 and 2,000 to 100,000).

[0286] In some embodiments, the one or more regions flanking the region encoding the target of interest can independently range in length from 15 to 1,000 nucleotides (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800 and 900 nucleotides or at least 30, 40, 45, 50 , 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 and 1,000 nucleotides).

[0287] In some embodiments, the mRNA includes a tailing sequence, which may range in length from none to 500 nucleotides (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450 or 500 nucleotides). If the tailing region is a polyA tail, the length may be determined in units of polyA binding protein binding or as a function of polyA binding protein binding. In this embodiment, the polyA tail is long enough to bind at least 4 monomers of the polyA binding protein. The polyA binding protein monomer binds to a stretch of approximately 38 nucleotides. Thus, it has been observed that polyA tails of approximately 80 nucleotides and 160 nucleotides are functional.

[0288] In some embodiments, the mRNA includes a capping sequence that includes a single cap or a series of nucleotides that form a cap. The capping sequence may be 1 to 10 nucleotides in length, such as 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or less nucleotides. In some embodiments, the capping sequence is absent.

[0289] In some embodiments, the mRNA includes a region containing an initiation codon. The length of the region containing the initiation codon may range from 3 to 40, such as 5-30, 10-20, 15, or at least 4 or 30 or less nucleotides.

[0290] In some embodiments, the mRNA includes a region containing a stop codon. The length of the region containing the stop codon may range from 3 to 40, such as 5-30, 10-20, 15, or at least 4 or 30 or less nucleotides.

[0291] In some embodiments, the mRNA includes regions containing restriction sequences. The length of the region containing the restriction sequence may range from 3 to 40, such as 5-30, 10-20, 15, or at least 4 or 30 or less nucleotides. [Untranslated area] [(] [UTR] [)]

[0292] In some embodiments, the mRNA includes at least one untranslated region (UTR) flanking a region encoding a target of interest. The UTR is transcribed, but not translated.

[0293] The 5' UTR starts at the transcription start site and continues to the start codon, but does not include the start codon; while the 3' UTR starts at the stop codon and continues until the transcription termination signal. While not wishing to be bound by theory, UTRs may have a regulatory role in the translation of nucleic acids and their stability.

[0294] Native 5' UTRs often include features that have a role in translation initiation, as they tend to include Kozak sequences that are commonly known to be involved in the process by which ribosomes initiate translation of many genes. The Kozak sequence has the common CCR(A / G)CCAUGG, where R is the purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G". The 5'UTR is also known to form secondary structures involved in elongation factor binding.

[0295] It is known that the 3' UTR has extensions of adenosine and uridine embedded in it. Such AU-rich tags are particularly prevalent in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be divided into three categories (Chen et al., 1995): Class I AREs contain several scattered copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain class I AREs. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of ARE include GM-CSF and TNF-a. Class III AREs are less clearly defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myogenin are two well-studied examples of this category. Most proteins that bind to the ARE are known to destabilize the message, while members of the ELAV family (most notably, HuR) have been documented to increase mRNA stability. HuR binds to all three classes of AREs. Engineering HuR-specific binding sites into the 3' UTR of nucleic acid molecules will cause HuR binding and, therefore, the stabilization of messages in vivo. The introduction, removal, or modification of 3' UTR AU-rich elements (AREs) can be used to modulate mRNA stability. For example, one or more copies of an ARE can be introduced to render the mRNA less stable and therefore reduce translation and reduce the yield of the resulting protein. Alternatively, an ARE can be identified and removed or mutated to increase intracellular stability, and thus increase translation and production of the resulting protein.

[0296] In some embodiments, introducing features commonly expressed in genes of target organs can enhance the stability of mRNA and protein production in specific organs and / or tissues. As a non-limiting example, the characteristic may be a UTR. As another example, the feature may be an intron or part of an intron sequence. [5'] [Cap]

[0297] The 5' cap structure of mRNA participates in nuclear export, increases mRNA stability, and binds to mRNA cap-binding protein (CBP), which forms a mature circular mRNA species through the combination of CBP and poly(A)-binding protein and is responsible for the stability of mRNA in cells. and translation ability. The cap further facilitates removal of the 5' proximal intron during mRNA splicing.

[0298] Endogenous mRNA molecules can be capped at the 5' end, resulting in a 5'-ppp-5'-triphosphate bond between the terminal guanosine cap residue of the mRNA molecule and the transcribed sense nucleotide at the 5' end. Union. This 5'-guanylate cap can then be methylated to produce an N7-methyl-guanylate residue. The ribose sugar of the 5' end of the mRNA and / or the anteterminal transcribed nucleotide may also be 2'-O-methylated as appropriate. 5'-decapping via hydrolysis and cleavage of the guanylate cap structure can target nucleic acid molecules, such as mRNA molecules, for degradation.

[0299] Modifications to the mRNA can create non-hydrolyzable cap structures that prevent decapping and therefore increase the half-life of the mRNA. Since hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphodiester linkage, modified nucleotides can be used during the capping reaction. For example, vaccinia capping enzyme from New England Biolabs (Ipswich, MA) and a-thio-guanosine nucleotide can be used according to the manufacturer's instructions to generate thio in the 5'-ppp-5' cap. Phosphate linkage.

[0300] Additional modified guanosine nucleotides such as alpha-methyl-phosphonate and seleno-phosphate nucleotides can be used.

[0301] Additional modifications include, but are not limited to, 2'-O-methylation of the ribose sugar on the 2'-hydroxyl group of the sugar ring at the 5' end of the mRNA (as mentioned above) and / or the 5' end pre-nucleotide. A variety of different 5' cap structures can be used to generate 5' caps for nucleic acid molecules, such as mRNA molecules.

[0302] Cap analogs, also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, are chemically identical to natural (i.e., endogenous, wild-type, or physiological) cap analogs. ) 5' cap is different while retaining cap function. Cap analogs can be chemically (ie, non-enzymatic) or enzymatically synthesized and / or linked to nucleic acid molecules.

[0303] For example, the anti-reverse cap analogue (ARCA) cap contains two guanines linked by a 5'-5'-triphosphate group, one of which contains an N7 methyl group and a 3'-0-methyl group ( Namely, N7,3'-0-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m 7G-3'mppp-G; which can be equivalently designated as 3' O-Me -m7G(5')ppp(5')G). The 3'-0 atom of another unmodified guanine becomes linked to the 5' terminal nucleotide of a capped nucleic acid molecule (e.g., mRNA). N7- and 3'-O-methylated guanine provides the terminal portion for capping nucleic acid molecules (eg, mRNA).

[0304] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-O-methyl on guanosine (i.e., N7,2'-0-dimethyl-guanosine-5'-triphosphate- 5'-guanosine,m 7Gm-ppp-G).

[0305] Although cap analogs allow concomitant capping of nucleic acid molecules in in vitro transcription reactions, up to 20% of transcripts can remain uncapped. This, as well as structural differences between cap analogues and the endogenous 5' cap structure of nucleic acids produced by the endogenous cellular transcription machinery, can result in reduced translational capacity and reduced cellular stability.

[0306] The mRNA can also be capped post-transcriptionally using enzymes to produce a more reliable 5' cap structure. As used herein, the phrase "more reliable" refers to characteristics that closely mirror or mimic endogenous or wild-type characteristics, either structurally or functionally. That is, "more reliable" features better represent endogenous, wild-type, native or physiological cell function and / or structure, or one or more aspects, than prior art synthetic features or analogs, etc. Outperforms corresponding endogenous, wild-type, native or physiological characteristics. Non-limiting examples of more reliable 5' cap structures are, inter alia, those compared to synthetic 5' cap structures known in the art (or compared to wild-type, natural or physiological 5' cap structures). ) has enhanced binding of cap-binding proteins, increased half-life, reduced sensitivity to 5' endonucleases and / or reduced 5' decapping. For example, recombinant vaccinia virus capping enzyme and recombinant 2'-0-methyltransferase can generate a typical 5'-5'-tris between the 5' end nucleotide of the mRNA and the guanine cap nucleotide. Phosphate linkage, in which the cap guanine contains N7 methylation, and the 5' end nucleotide of the mRNA contains 2'-0-methyl. Such structures are called Capl structures. This cap results in higher translational capacity and cell stability as well as reduced activation of cellular pro-inflammatory cytokines compared to, for example, other 5' cap analog structures known in the art. Cap structures include but are not limited to 7mG(5*)ppp(5*)N,pN2p (cap0), 7mG(5*)ppp(5*)NlmpNp (cap1) and 7mG(5*)-ppp(5' )NlmpN2mp (cap 2).

[0307] In some embodiments, the 5' end cap may include an endogenous cap or a cap analog.

[0308] In some embodiments, the 5' end cap can comprise a guanine analog. Suitable guanine analogs include, but are not limited to, inosine, Nl-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-side oxy-guanosine, 2-amino-guanosine Guanosine, LNA-guanosine and 2-azido-guanosine. [IRES] [sequence]

[0309] In some embodiments, the mRNA can contain an internal ribosome entry site (IRES). First identified as a characteristic picornaviridae viral RNA, IRES play an important role in initiating protein synthesis in the absence of a 5' cap structure. An IRES can serve as the only ribosome binding site, or it can serve as one of multiple ribosome binding sites on the mRNA. An mRNA containing more than one functional ribosome binding site may encode several peptides or polypeptides that are independently translated by ribosomes. Non-limiting examples of IRES sequences that can be used include, but are not limited to, those from picornaviruses (e.g., FMDV), pest viruses (CFFV), poliovirus (PV), encephalomyocarditis virus (ECMV) ), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukemia virus (MLV), simian immunodeficiency virus (SIV) or cricket paralysis virus (CrPV). [Gather] [A] [Tail]

[0310] During RNA processing, long chains of adenine nucleotides (poly-A tails) can be added to polynucleotides such as mRNA molecules to increase stability. Immediately following transcription, the 3' end of the transcript can be cleaved to release the 3' hydroxyl group. Next, poly(A) polymerase adds a series of adenine nucleotides to RA. This process, called polyadenylation, adds a polyA tail of a certain length.

[0311] In some embodiments, the polyA tail is greater than 30 nucleotides in length. In another embodiment, the polyA tail length is greater than 35 nucleotides (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500 and 3 ,000 nucleotides). In some embodiments, the mRNA includes about 30 to about 3,000 nucleotides (e.g., 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 750, 30 to 1,000, 30 to 1,500, 30 to 2,000 , 30 to 2,500, 50 to 100, 50 to 250, 50 to 500, 50 to 750, 50 to 1,000, 50 to 1,500, 50 to 2,000, 50 to 2,500, 50 to 3,000, 100 to 500, 100 to 750 , 100 to 1,000, 100 to 1,500, 100 to 2,000, 100 to 2,500, 100 to 3,000, 500 to 750, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 2,500, 500 to 3,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 2,500, 1,000 to 3,000, 1,500 to 2,000, 1,500 to 2,500, 1,500 to 3,000, 2,000 to 3,000, 2,000 to 2,500 and 2,500 to 3,000).

[0312] In some embodiments, the poly-A tail is designed relative to the length of the entire mRNA. This design may be based on the length of the region encoding the target of interest, the length of specific features or regions such as flanking regions, or the length of the final product expressed from the mRNA.

[0313] In this context, the length of the polyA tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% longer than the mRNA or its characteristics. The poly A tail can also be designed as part of the mRNA to which it belongs. In this context, the poly-A tail may be the full length of the construct or the full length of the construct minus 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the poly-A tail. In addition, the engineered binding sites and mRNA binding of poly-A binding proteins can enhance performance.

[0314] In addition, multiple different mRNAs can be linked together via the 3' end of the poly A tail with PABP (poly A binding protein) using modified nucleotides. Transfection experiments can be performed in relevant cell lines, and protein production can be analyzed by ELISA at 12 hours, 24 hours, 48 ​​hours, 72 hours and 7 days after transfection.

[0315] In some embodiments, the mRNA is designed to include poly A-G quartets. A G-quartet is a cyclic hydrogen-bonded array of four guanine nucleotides, which can be formed from G-rich sequences in DNA and RNA. In this example, a G quartet is incorporated at the end of the polyA tail. [Stop codon]

[0316] In some embodiments, the mRNA may include a stop codon. In some embodiments, the mRNA can include two stop codons. In some embodiments, the mRNA can include three stop codons. In some embodiments, the mRNA can include at least one stop codon. In some embodiments, the mRNA can include at least two stop codons. In some embodiments, the mRNA can include at least three stop codons. As a non-limiting example, the stop codon can be selected from TGA, TAA and TAG.

[0317] In some embodiments, the mRNA includes the stop codon TGA and an additional stop codon. In another embodiment, the added stop codon can be TAA. [ring] [RNA] [(oRNA)]

[0318] In some embodiments, the initial construct and / or the reference construct is circular RNA (oRNA). As used herein, the terms "oRNA" or "circular RNA" are used interchangeably and may refer to RNA that forms a circular structure via covalent or non-covalent bonds.

[0319] In some embodiments, the oRNA can be non-immunogenic in mammals (e.g., humans, non-human primates, rabbits, rats, and mice).

[0320] In some embodiments, the oRNA is capable of replicating or replicable in cells from aquatic animals (e.g., fish, crabs, shrimps, oysters, etc.), mammalian cells, from pet or zoo animals (e.g., cats, dogs, lizards, etc.) , birds, lions, tigers and bears, etc.), cells from agricultural or draft animals (such as horses, cows, pigs, chickens, etc.), human cells, cultured cells, primary cells or cell lines, stem cells, precursors cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells (e.g., normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any combination thereof .

[0321] In some embodiments, the oRNA has a half-life that is at least that of the linear counterpart. In some embodiments, the oRNA has an increased half-life relative to the half-life of its linear counterpart. In some embodiments, the half-life is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or greater. In some embodiments, the oRNA has a half-life or persistence in the cell from at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days or longer or any time in between. In some embodiments, the half-life or persistence of the oRNA in the cell is no more than about 10 minutes to about 7 days, or no more than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours (1 day ), 36 hours (1.5 days), 48 hours (2 days), 60 hours (2.5 days), 72 hours (3 days), 4 days, 5 days, 6 days or 7 days.

[0322] In some embodiments, the oRNA has a half-life or persistence in the cell while the cell is dividing. In some embodiments, the oRNA has a half-life or persistence in postmitotic cells. In certain embodiments, the oRNA has a half-life or persistence in dividing cells from greater than about 10 minutes to about 30 days, or at least about 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 hours (1 day ), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days , 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days or longer or any time in between .

[0323] In some embodiments, oRNA modulates cellular function, e.g., temporarily or long-term. In certain embodiments, stably alters cellular function, such as maintaining regulation for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days or longer. In certain embodiments, temporarily altering cellular function, such as maintaining regulation for no more than about 30 minutes to about 7 days, or no more than about 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours , 23 hours, 24 hours (1 day), 36 hours (1.5 days), 48 hours (2 days), 60 hours (2.5 days), 72 hours (3 days), 4 days, 5 days, 6 days or 7 days .

[0324] In some embodiments, the oRNA is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides Nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleosides acid, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, At least about 19,000 nucleotides or at least about 20,000 nucleotides. In some embodiments, the oRNA may be of sufficient size to accommodate a ribosome binding site.

[0325] In some embodiments, the maximum size of the oRNA may be limited by the ability to encapsulate and deliver the RNA to the target. In some embodiments, the oRNA is of sufficient length to encode a suitable polypeptide, and is therefore at least 20,000 nucleotides, at least 15,000 nucleotides, at least 10,000 nucleotides, at least 7,500 nucleotides, or at least 5,000 nucleotides. nucleotides, at least 4,000 nucleotides, at least 3,000 nucleotides, at least 2,000 nucleotides, at least 1,000 nucleotides, at least 500 nucleotides, at least t 400 nucleotides, at least 300 Lengths of nucleotides, at least 200 nucleotides, at least 100 nucleotides may be suitable.

[0326] In some embodiments, the oRNA includes one or more elements described elsewhere herein. In some embodiments, elements may be separated from each other by spacer sequences or linkers. In some embodiments, the elements can be separated from each other by 1 nucleotide, 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides. nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 80 nucleotides, about 100 nucleotides, about 150 nucleosides Acid, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides , about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, up to about 1 kb, at least about 1000 nucleotides.

[0327] In some embodiments, one or more elements are adjacent to each other, such as lacking spacing elements.

[0328] In some embodiments, one or more elements are flexible in configuration. In some embodiments, conformational flexibility is due to the sequence being substantially free of secondary structure.

[0329] In some embodiments, the oRNA contains secondary or tertiary structure that accommodates binding sites for ribosome, translation, or cyclic translation.

[0330] In some embodiments, an oRNA contains specific sequence characteristics. For example, an oRNA can contain a specific nucleotide composition. In some such embodiments, the oRNA may include one or more purine-rich regions (adenine or guanosine). In some such embodiments, the oRNA may include one or more purine-rich regions (adenine or guanosine). In some embodiments, an oRNA may include one or more AU-rich regions or elements (AREs). In some embodiments, an oRNA can include one or more adenine-rich regions.

[0331] In some embodiments, the oRNA contains one or more modifications described elsewhere herein.

[0332] In some embodiments, an oRNA includes one or more expression sequences and is configured for sustained expression in cells within an individual's body. In some embodiments, an oRNA is configured such that expression of one or more expression sequences in the cell at a later time point is equal to or greater than at an earlier time point. In such embodiments, the performance of one or more performance sequences may remain at a relatively stable level or may increase over time. The performance of the performance sequence can be relatively stable for an extended period of time. For example, in some cases, the expression of one or more expression sequences in a cell is inconsistent for a period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days. Reduce by 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5%. In some cases, the expression of one or more expression sequences in cells remains unchanged by no more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, The 10% or 5% level remains for at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 days or longer. [Adjustment element]

[0333] In some embodiments, the oRNA contains regulatory elements. As used herein, a "modulating element" is a sequence that modulates the performance of a sequence of expressions. Adjustment elements may include sequences located adjacent the payload or cargo area. The adjustment element may be operatively connected to the payload or load area.

[0334] In some embodiments, an adjustment element may increase the amount of payload or load expressed compared to the amount expressed when no adjustment element is present. As a non-limiting example, an adjustment element may increase the amount of payload or load represented by a sequence of multiple payloads or loads connected in series.

[0335] In some embodiments, a regulatory element may comprise a sequence that selectively initiates or activates translation of the payload or cargo.

[0336] In some embodiments, the regulatory element may comprise a sequence that initiates degradation of the oRNA or payload or carrier. Non-limiting examples of sequences that initiate degradation include, but are not limited to, riboswitch aptazymes and miRNA binding sites.

[0337] In some embodiments, regulatory elements may regulate translation of payloads or cargoes in oRNAs. Modulation can result in an increase (enhancer) or decrease (repressor) in the payload or cargo. The adjustment element may be located adjacent to the payload or load (eg, on one or both sides of the payload or load).

[0338] In some embodiments, the translation initiation sequence serves as a regulatory element. In some embodiments, the translation initiation sequence includes AUG / ATG codons. In some embodiments, the translation initiation sequence includes any eukaryotic initiation codon, such as, but not limited to, AUG / ATG, CUG / CTG, GUG / GTG, UUG / TTG, ACG, AUC / ATC, AUU, AAG, AUA / ATA or AGG. In some embodiments, the translation initiation sequence includes a Kozak sequence. In some embodiments, translation begins with an alternative translation initiation sequence, e.g., a translation initiation sequence other than AUG / ATG, under selective conditions (e.g., stress-inducing conditions). As a non-limiting example, translation of cyclic polyribonucleotides can begin with an alternative translation initiation sequence, such as ACG. As another non-limiting example, cyclic polyribonucleotide translation can begin with the alternative translation initiation sequence CUG / CTG. As another non-limiting example, translation can begin with the alternative translation initiation sequence GUG / GTG. As yet another non-limiting example, translation can begin with a repeat-related non-AUG (RAN) sequence, such as an alternative translation initiation sequence that includes a short stretch of the repetitive RNA (e.g., CGG, GGGGCC, CAG, CTG). [Masking agent]

[0339] Masking any nucleotide flanking the codon that initiates translation can be used to alter the translation initiation position, translation efficiency, length and / or structure of the oRNA. In some embodiments, a masking agent can be used near an initiation codon or an alternative initiation codon to mask or conceal the codon to reduce the risk of translation start probability. Non-limiting examples of masking agents include antisense locked nucleic acid (LNA) oligonucleotides and exon junction complexes (EJC). In some embodiments, a masking agent can be used to mask the initiation codon of an oRNA to increase the likelihood that translation will initiate at an alternative initiation codon. [Translation start sequence]

[0340] In some embodiments, an oRNA encodes a polypeptide or peptide and may include a translation initiation sequence. The translation initiation sequence may include, but is not limited to, an initiation codon, a noncoding initiation codon, a Kozak sequence, or a Shine-Dalgarno sequence. The translation initiation sequence may be located adjacent to the payload or payload (eg, on one or both sides of the payload or payload).

[0341] In some embodiments, the translation initiation sequence provides conformational flexibility to the oRNA. In some embodiments, the translation initiation sequence is within a substantially single-stranded region of the oRNA.

[0342] The oRNA may include more than 1 start codon, such as, but not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13 , at least 14, at least 15 or more than 15 start codons. Translation can initiate on the first initiation codon or can initiate downstream of the first initiation codon.

[0343] In some embodiments, the oRNA can initiate at a codon that is not the first initiation codon (eg, AUG). Translation of cyclic polyribonucleotides can be initiated by alternative translation initiation sequences, such as, but not limited to, ACG, AGG, AAG, CUG / CTG, GUG / GTG, AUA / ATA, AUU / ATT, UUG / TTG. In some embodiments, translation begins with an alternative translation initiation sequence under selective conditions, such as stress-inducing conditions. As a non-limiting example, translation of an oRNA can begin with an alternative translation initiation sequence, such as ACG. As another non-limiting example, oRNA translation can begin with the alternative translation initiation sequence CUG / CTG. As yet another non-limiting example, oRNA translation can begin with the alternative translation initiation sequence GTG / GUG. As yet another non-limiting example, the oRNA can initiate translation at a repeat-related non-AUG (RAN) sequence, such as an alternative translation initiation sequence that includes a short stretch of the repetitive RNA (e.g., CGG, GGGGCC, CAG, CTG) . [IRES] [sequence]

[0344] In some embodiments, oRNAs described herein comprise an internal ribosome entry site (IRES) element capable of engaging eukaryotic ribosomes. In some embodiments, an IRES element is at least about 5 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least About 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 250 nucleotides, at least about 350 nucleotides, or at least about 500 nucleotides. In one embodiment, the IRES element is derived from the DNA of organisms including, but not limited to, viruses, mammals, and Drosophila. Such viral DNA can be derived from, but is not limited to, picornavirus complementary DNA (cDNA), encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA. In one embodiment, the Drosophila DNA from which the IRES element is derived includes, but is not limited to, the antennapedia gene from Drosophila melanogaster.

[0345] In some embodiments, the IRES element is derived at least in part from a virus, for example it can be derived from a viral IRES element such as ABPV_IGRpred, AEV, ALPV_IGRpred, BQCV_IGRpred, BVDV1_1-385, BVDV1_29-391, CrPV_5NCR, CrPV_IGR, crTMV_IREScp, crTMV_IRESmp75, crTMV_IRESmp228 , crTMV_IREScp, crTMV_IREScp, CSFV, CVB3, DCV_IGR, EMCV-R, EoPV_5NTR, ERAV 245-961, ERBV 162-920, EV71_1-748, FeLV-Notch2, FMDV_type_C, GBV-A, GBV-B, GBV-C, gypsy_env , gypsyD5, gypsyD2, HAV_HM175, HCV_type_1a, HiPV_IGRpred, HIV-1, HoCV1_IGRpred, HRV-2, IAPV_IGRpred, idefix, KBV_IGRpred, LINE-1_ORF1_-101_to_-1, LINE-1_ORF1-302_to_-202, LINE-1_ORF2-13 8_to_-86 , LINE-1_ORF1_-44to_-1, PSIV_IGR, PV_type1_Mahoney, PV_type3_Leon, REV-A, RhPV_5NCR, RhPV_IGR, SINV1_IGRpred, SV40_661-830, TMEV, TMV_UI_IRESmp228, TRV_5NTR, TrV_IGR, or TSV_IGR. In some embodiments, the IRES element is derived at least in part from a cellular IRES, such as AML1 / RUNX1, Antp-D, Antp-DE, Antp-CDE, Apaf-1, Apaf-1, AQP4, AT1R_var1, AT1R_var2, AT1R_var3, AT1R_var4 , BAG1_p36delta236 nt, BAG1_p36, BCL2, BiP_-222_-3, c-IAP1_285-1399, c-IAP1_1313-1462, c-jun, c-myc, Cat-1224, CCND1, DAPS, eIF4G, eIF4GI-ext, eIF4GII, eIF4GII-long, ELG1, ELH, FGF1A, FMR1, Gtx-133-141, Gtx-1-166, Gtx-1-120, Gtx-1-196, hairless, HAP4, HIF1a, hSNM1, Hsp101, hsp70, hsp70, Hsp90, IGF2_leader2, Kv1.4_1.2, L-myc, LamB1_-335_-1, LEF1, MNT_75-267, MNT_36-160, MTG8a, MYB, MYT2_997-1152, n-MYC, NDST1, NDST2, NDST3, NDST4L, NDST4S, NRF_-653_-17, NtHSF1, ODC1, p27kip1, 03_128-269, PDGF2 / c-sis, Pim-1, PITSLRE_p58, Rbm3, reaper, Scamper, TFIID, TIF4631, Ubx_1-966, Ubx_373- 961, UNR, Ure2, UtrA, VEGF-A-133-1, XIAP_5-464, XIAP_305-466 or YAP1. [terminating element]

[0346] In some embodiments, an oRNA includes one or more payload or payload sequences (also known as expression sequences), and each payload or payload sequence may or may not have a terminating element.

[0347] In some embodiments, the oRNA includes one or more carrier or payload sequences, and the sequences lack termination elements to allow continued translation of the oRNA. Exclusion of the termination element may invert translation of the cycle or continuous expression of the encoded peptide or polypeptide, since the ribosomes will not stall or reduce. In such embodiments, rolling circle translation represents a continuous representation through each load or payload sequence.

[0348] In some embodiments, one or more of the carrier or payload sequences in the oRNA comprise a termination element.

[0349] In some embodiments, not all payload or payload sequences in the oRNA contain termination elements. In such cases, the cargo or payload can fall off the ribosome when it encounters the termination element and terminates translation. In some embodiments, translation is terminated while at least one region of the ribosome remains in contact with the oRNA. [Rolling Ring Translation]

[0350] In some embodiments, once translation of the oRNA is initiated, ribosomes bound to the oRNA do not dissociate from the oRNA until at least one round of oRNA translation is completed. In some embodiments, an oRNA as described herein is competent for rolling circle translation. In some embodiments, during rolling circle translation, upon completion of at least 2 rounds, at least 3 rounds, at least 4 rounds, at least 5 rounds, at least 6 rounds, at least 7 rounds, at least 8 rounds, At least 9 rounds, at least 10 rounds, at least 11 rounds, at least 12 rounds, at least 13 rounds, at least 14 rounds, at least 15 rounds, at least 20 rounds, at least 30 rounds, at least 40 rounds, at least 50 rounds, at least 60 rounds, at least 70 rounds rounds, at least 80 rounds, at least 90 rounds, at least 100 rounds, at least 150 rounds, at least 200 rounds, at least 250 rounds, at least 500 rounds, at least 1000 rounds, at least 1500 rounds, at least 2000 rounds, at least 5000 rounds, at least 10000 rounds, The ribosome bound to the oRNA will not detach from the oRNA until at least 10.sup.5 rounds or at least 10.sup.6 rounds of oRNA translation.

[0351] In some embodiments, rolling circle translation of an oRNA results in polypeptides that are translated from more than one round of oRNA translation. In some embodiments, the oRNA includes a stagger element, and rolling circle translation of the oRNA results in a polypeptide product resulting from a single round of oRNA translation or less than a single round of oRNA translation. [cyclization]

[0352] In one embodiment, linear RNA can be circularized or concatemerized. In some embodiments, linear RNA can be circularized in vitro prior to formulation and / or delivery. In some embodiments, linear RNA can be circularized within the cell.

[0353] In some embodiments, the mechanism of cyclization or concatenation can occur via at least 3 different pathways: 1) chemical, 2) enzymatic, and 3) ribonuclease catalyzed. The newly formed 5'- / 3'-linkages can be intramolecular or intermolecular.

[0354] In the first pathway, the 5' and 3' ends of the nucleic acid contain chemically reactive groups that, when brought together, form a new link between the 5' and 3' ends of the molecule. Covalent bonding. The 5' end can contain an NHS-ester reactive group, and the 3' end can contain a 3'-amine-terminated nucleotide, so that the 3'-end on the 3' end of the mRNA molecule can be synthesized in an organic solvent. The amine-terminated nucleotide will undergo nucleophilic attack on the 5'-NHS-ester moiety, thereby forming a new 5'- / 3'-amide bond.

[0355] In the second pathway, T4 RNA ligase can be used to enzymatically connect the 5'-phosphorylated nucleic acid molecule to the 3'-hydroxyl group of the nucleic acid, thereby forming a new phosphodiester linkage. In one example reaction, 2 g of nucleic acid molecules were incubated with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) at 37°C for 1 hour according to the manufacturer's protocol. The ligation reaction can be performed in the presence of a resolving oligonucleotide capable of base pairing with both the 5'- and 3'-regions of the ligation to assist in the enzymatic ligation reaction.

[0356] In the third approach, the 5' end or 3' end of the cDNA template encodes a ligase ribonuclease sequence, so that during in vitro transcription, the resulting nucleic acid molecule can contain a ligase capable of joining the 5' end of the nucleic acid molecule and the 3' end of the nucleic acid molecule. ' end of the active ribonuclease sequence. Ligase ribonucleases can be derived from group I introns, group I introns, hepatitis D virus, hairpin ribonucleases, or can be derived from systematic evolution of ligands by exponential enrichment; SELEX) to choose. The RNase ligase reaction can take 1 to 24 hours at temperatures between 0 and 37°C.

[0357] In some embodiments, oRNA is produced by circularization of linear RNA. [Extracellular cyclization]

[0358] In some embodiments, linear RNA is circularized or concatenated using chemical methods to form oRNA. In some chemical methods, the 5' and 3' ends of a nucleic acid (e.g., linear RNA) include chemically reactive groups that, when brought into close proximity, can bind to each other at the 5' and 3' ends of the molecule. New covalent bonds are formed between the ends. The 5' end can contain an NHS-ester reactive group, and the 3' end can contain a 3'-amine-terminated nucleotide, so that in organic solvents, the 3'-amine on the 3' end of the linear RNA The base-capped nucleotide will undergo nucleophilic attack on the 5'-NHS-ester moiety, thereby forming a new 5'- / 3'-amide bond.

[0359] In one embodiment, a DNA or RNA ligase can be used to enzymatically connect a 5'-phosphorylated nucleic acid molecule (e.g., linear RNA) to the 3'-hydroxyl group of the nucleic acid (e.g., linear nucleic acid), thereby forming a new phosphodiester linkage . In one example reaction, linear RNA was incubated with 1-10 units of T4 RNA ligase at 37°C for 1 hour according to the manufacturer's protocol. The ligation reaction can be performed in the presence of a linear nucleic acid capable of base pairing with both the 5'- and 3'-regions of the ligation to assist in the enzymatic ligation reaction. In one embodiment, the ligation is a splint ligation, in which a single-stranded polynucleotide (splint), such as a single-stranded RNA, can be designed to hybridize to both ends of a linear RNA such that both ends can hybridize to the single-stranded splint. parallel connection. Therefore, splint ligase can catalyze the joining of two juxtaposed ends of linear RNA to generate oRNA.

[0360] In one embodiment, DNA or RNA ligase can be used to synthesize oRNA. As a non-limiting example, the ligase may be a circ ligase or a circ ligase.

[0361] In one embodiment, the 5' or 3' end of the linear RNA may encode a ligase ribonuclease sequence such that during in vitro transcription, the resulting linear RNA includes a ligase capable of joining the 5' end of the linear RNA to the 3' end of the linear RNA. The active ribonuclease sequence at the end. Ligase ribonucleases can be derived from group I introns, hepatitis D virus, hairpin ribonucleases, or can be selected by systematic evolution of ligands by exponential enrichment (SELEX).

[0362] In one embodiment, linear RNA can be circularized or concatenated by using at least one non-nucleic acid moiety. In one aspect, the at least one non-nucleic acid moiety can react with regions or features near the 5' end and / or near the 3' end of the linear RNA to circularize or concatenate the linear RNA. In another aspect, the at least one non-nucleic acid portion can be located in, linked to or near the 5' end and / or the 3' end of the linear RNA. The non-nucleic acid moieties contemplated may be homologous or heterologous. As non-limiting examples, the non-nucleic acid moiety may be a linkage, such as a hydrophobic linkage, an ionic linkage, a biodegradable linkage, and / or a cleavable linkage. As another non-limiting example, the non-nucleic acid moiety is a conjugating moiety. As yet another non-limiting example, the non-nucleic acid moiety may be an oligonucleotide or peptide moiety, such as an aptamer or non-nucleic acid linker as described herein.

[0363] In one embodiment, linear RNA can be cyclized or concatenated due to non-nucleic acid moieties that cause attraction between the 5' and 3' ends of the linear RNA, near the atoms or surfaces of the molecule to which it is attached. As a non-limiting example, one or more linear RNAs can be circularized or concatenated by intermolecular or intramolecular forces. Non-limiting examples of intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, Van der Waals forces, and London dispersion forces. . Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonant bonds, agnostic bonds, dipolar bonds, conjugation, hyperconjugation, and anti-bonding.

[0364] In one embodiment, the linear RNA can comprise a ribonuclease RNA sequence near the 5' end and near the 3' end. The ribonuclease RNA sequence can be covalently linked to the peptide when the sequence is exposed to the remainder of the ribonuclease. In one aspect, peptides covalently linked to ribonuclease RNA sequences near the 5' and 3' ends can bind to each other, thereby circularizing or concatenating the linear RNA. In another aspect, the peptide covalently linked to the ribonuclease RNA near the 5' and 3' ends can be Circularize or concatenate linear RNA.

[0365] In some embodiments, linear RNA can include nucleic acids that are converted to 5' monophosphates, for example, by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or ATP diphosphohydrolase (apyrase) of 5' triphosphate. Alternatively, the conversion of the 5' triphosphate of the linear RNA into the 5' monophosphate can be performed by a two-step reaction involving the following: (a) reacting the 5' nucleotide of the linear RNA with a phosphatase (e.g., Antarctic phosphatase, contact shrimp alkaline phosphatase or calf intestinal phosphatase) to remove all three phosphates; and (b) contact the 5' nucleotide after step (a) with a kinase that adds a single phosphate (e.g., poly nucleotide kinase) contact.

[0366] In some embodiments, RNA can be cyclized using methods described in WO2017222911 and WO2016197121, the contents of each of which are incorporated herein by reference in their entirety.

[0367] In some embodiments, the RNA can be circularized, for example, by back-splicing non-mammalian exogenous introns or splinting the 5' and 3' ends of the linear RNA. In one embodiment, a circular RNA is produced from a recombinant nucleic acid encoding a target RNA to be prepared as a circular form. As a non-limiting example, the method includes: a) generating a recombinant nucleic acid encoding a target RNA to be prepared as a circular form, wherein the recombinant nucleic acid includes in 5' to 3' order: i) includes exogenous 3' splice site The 3' portion of the intron, ii) the nucleic acid sequence encoding the target RNA, and iii) the 5' portion of the exogenous intron including the 5' splice site; b) undergoes transcription such that the RNA is produced from the recombinant nucleic acid; and c) perform RNA splicing, resulting in RNA circularization to produce oRNA.

[0368] While not wishing to be bound by theory, the circRNAs produced with exogenous introns are recognized by the immune system as "non-self" and trigger an innate immune response. On the other hand, the circRNAs produced with endogenous introns are recognized by the immune system as "self" and generally do not stimulate innate immune responses, even if they carry exons containing foreign RNA.

[0369] Therefore, if desired, circular RNAs with endogenous or exogenous introns can be generated to control immunological self / non-self differentiation. A variety of intron sequences are known from a wide variety of organisms and viruses, and include sequences derived from genes encoding proteins, ribosomal RNA (rRNA), or transfer RNA (tRNA).

[0370] Circular RNA can be produced from linear RNA in a variety of ways. In some embodiments, circular RNA is generated from linear RNA by back-splicing the downstream 5' splice site (splice donor) to the upstream 3' splice site (splice acceptor). Circular RNAs can be produced in this manner by any non-mammalian splicing method. For example, linear RNA containing various types of introns, including self-splicing group I introns, self-splicing group II introns, spliceosomal introns, and tRNA introns, can be circularized. In particular, group I and group II introns are advantageous because they can be readily used to generate circular RNAs in vitro as well as in vivo due to their ability to self-splicing due to their autocatalytic ribonuclease activity.

[0371] In some embodiments, circular RNA can be generated in vitro from linear RNA by chemically or enzymatically joining the 5' and 3' ends of the RNA. Chemical conjugation can, for example, use cyanogen bromide (BrCN) or ethyl-3-(3'-dimethylaminopropyl)carbodiimide (EDC) for activation of the nucleotide phosphate monoester group to allow phosphobis Ester bond formation occurs. See, for example, Sokolova (1988) FEBS Lett 232: 153-155; Dolinnaya et al. (1991) Nucleic Acids Res., 19:3067-3072; Fedorova (1996) Nucleosides Nucleotides Nucleic Acids 15: 1 137-1 147; which are cited in are incorporated into this article. Alternatively, enzymatic conjugation can be used to circularize RNA. Exemplary ligases that can be used include T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl 1), and T4 RNA ligase 2 (T4 Rnl 2).

[0372] In some embodiments, splint ligation using oligonucleotide splints that hybridize to both ends of the linear RNA can be used to join the ends of the linear RNA together. Hybridization of the splint (which can be DNA or RNA) orients the 5'-phosphate and 3'-OH of the RNA termini for ligation. Subsequent conjugation can be performed using chemical or enzymatic techniques, as described above. Enzymatic ligation can be performed, for example, with T4 DNA ligase (required for DNA splints), T4 RNA ligase 1 (required for RNA splints) or T4 RNA ligase 2 (DNA or RNA splints). In some cases, chemical ligation, such as with BrCN or EDC, may be more efficient than enzymatic ligation if the structure of the hybridized splint-RNA complex interferes with enzymatic activity.

[0373] In some embodiments, the oRNA can further comprise an internal ribosome entry site (IRES) operably linked to the RNA sequence encoding the polypeptide. Inclusion of an IRES permits translation of one or more open reading frames from the circular RNA. IRES elements attract eukaryotic ribosomal translation initiation complexes and promote translation initiation. See, for example, Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20 : 102-110; Kobayashi et al., BioTechniques (1996) 21: 399-402; and Mosser et al., BioTechniques 1997 22 150-161).

[0374] In some embodiments, the cyclization methods provided herein have a cyclization efficiency of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40 %, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or 100%. In some embodiments, the cyclization methods provided herein have a cyclization efficiency of at least about 40%. [splicing element]

[0375] In some embodiments, the oRNA includes at least one splicing element. The splicing element can be a complete splicing element that can mediate splicing of the oRNA, or the splicing element can be a residual splicing element from a completed splicing event. For example, in some cases, splicing elements of a linear RNA may mediate splicing events that cause linear RNA circularization, such that the resulting oRNA contains residual splicing elements from such splicing-mediated circularization events. In some cases, residual splicing elements are unable to mediate any splicing. In other cases residual splicing elements may still mediate splicing under certain circumstances. In some embodiments, the splicing element is adjacent to at least one expression sequence. In some embodiments, the oRNA includes splicing elements adjacent to each expressed sequence. In some embodiments, splicing elements are on one or both sides of each expression sequence, causing separation of expression products, such as peptides and / or polypeptides.

[0376] In some embodiments, the oRNA includes internal splicing elements where the splice ends are joined together upon replication. Some examples may include mini-introns (<100 nt) with splice site sequences and short inverted repeats (30-40 nt) (such as AluSq2, AluJr, and AluSz), inverted sequences flanking the intron , Alu elements in flanking introns, and motifs found in cis sequence elements close to back-splicing events (Table 4 enriched motifs above), such as at back-splice sites flanking exons Sequence in the 200 bp before (upstream) or after (downstream). In some embodiments, the oRNA includes at least one repetitive nucleotide sequence described elsewhere herein as an internal splicing element. In such embodiments, the repeating nucleotide sequence may include repeating sequences from Alu family introns. See, for example, US Patent No. 11,058,706.

[0377] In some embodiments, the oRNA can include typical splice sites flanking head-to-tail junctions of the oRNA.

[0378] In some embodiments, an oRNA can include a knob-helix-knob motif, which includes a 4 base pair stem flanked by two 3 nucleotide spikes. Cleavage occurs at a site in the ridge region, resulting in a characteristic fragment with a terminal 5'-hydroxyl group and a 2',3'-cyclic phosphate. Cyclization proceeds by nucleophilic attack of the 5'-OH group onto the 2',3'-cyclic phosphate of the same molecule, thereby forming a 3',5'-phosphodiester bridge.

[0379] In some embodiments, an oRNA can include sequences that mediate self-ligation. Non-limiting examples of sequences that may mediate self-ligation include autocyclizing introns (e.g., 5' and 3' slice junctions) or autocyclizing catalytic introns (such as group I, group II, or Group III introns). Non-limiting examples of Group I intron self-splicing sequences may include self-spliced ​​aligned intron-exon sequences derived from the T4 phage gene td, and the Tetrahymena intermediate sequence (IVS) rRNA. [Other cyclization methods]

[0380] In some embodiments, linear RNA can include complementary sequences, including repetitive or non-repetitive nucleic acid sequences within individual introns or throughout flanking introns. In some embodiments, oRNAs include repetitive nucleic acid sequences. In some embodiments, the repetitive nucleotide sequence includes a poly-CA or poly-UG sequence. In some embodiments, the oRNA includes at least one repeating nucleic acid sequence that hybridizes to a complementary repeating nucleic acid sequence in another segment of the oRNA, wherein the hybridized segment forms an internal double strand. In some embodiments, repeating nucleic acid sequences and complementary repeating nucleic acid sequences from two separate oRNAs hybridize to produce a single oRNA, with the hybridized segments forming an internal double strand. In some embodiments, complementary sequences are present at the 5' and 3' ends of the linear RNA. In some embodiments, the complementary sequences include about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more pairs Nucleotides.

[0381] In some embodiments, cyclization chemistries can be used to generate oRNA. Such methods may include, but are not limited to, click chemistry (e.g., alkyne- and azide-based methods or clickable bases), olefin metathesis, aminophosphate conjugation, semiamine aldehyde-imine cross-linking, base modification, and the like. Any combination.

[0382] In some embodiments, cyclization enzymatic methods can be used to generate oRNA. In some embodiments, ligases, such as DNA or RNA ligases, can be used to generate templates for oRNA or complementary sequences, complementary strands of oRNA, or oRNA. [Small distraction] [RNA (siRNA)]

[0383] In some embodiments, the payload region can be or encode an RNA interference (RNAi) sequence that can be used to reduce or inhibit gene expression. RNAi (also known as post-transcriptional gene silencing (PTGS), suppression or co-suppression) is a method of post-transcriptional gene silencing in which RNA molecules reduce or inhibit gene expression in a sequence-specific manner, often by damaging specific mRNA molecules. The active component of RNAi is short / small double-stranded RNA (dsRNA), called small interfering RNA (siRNA), which usually contains 15-30 nucleotides (for example, 19-25, 19-24 or 19-21 nucleotide) and 2 nucleotide 3' overhangs, and they match the nucleic acid sequence of the target gene. These short RNA species are naturally produced in vivo by Dicer-mediated cleavage of larger dsRNA and are functional in mammalian cells.

[0384] Naturally occurring small RNA molecules called microRNAs (miRNAs) trigger gene silencing by regulating the expression of mRNA. The RNA-induced silencing complex (RISC) containing miRNA targets nucleotides 2-7 in the 5' region of the miRNA (which is called the seed region) and exhibits perfect sequence complementarity with other base pairs in its 3' region. of mRNA. miRNA-mediated down-regulation of gene expression may be caused by cleavage of the target mRNA, translational inhibition of the target mRNA, or mRNA breakdown. The miRNA targeting sequence is usually located in the 3'-UTR of the target mRNA. A single miRNA can target more than 100 transcripts from various genes, and one mRNA can be targeted by different miRNAs.

[0385] siRNA duplexes or dsRNA targeting specific mRNAs can be designed and synthesized in vitro and introduced into cells to activate the RNAi process. It has previously been shown that 21-nucleotide siRNA duplexes, known as small interfering RNAs, can achieve potent and specific gene attenuation in mammalian cells without inducing an immune response. Post-transcriptional gene silencing by siRNA is rapidly emerging as a powerful tool for genetic analysis in mammalian cells, with the potential to generate new therapeutic agents.

[0386] siRNA sequences synthesized in vitro can be introduced into cells to activate RNAi. When introduced into a cell, similar to endogenous dsRNA, exogenous siRNA duplexes can assemble to form an RNA-induced silencing complex (RISC), an RNA sequence that interacts with one of the two strands of the siRNA duplex. or complementary (i.e. antisense)) interacting multi-unit complex. During this process, the siRNA's sense strand (or passenger strand) is lost from the complex, while the siRNA's antisense strand (or guide strand) matches its complementary RNA. Specifically, siRNAs containing RISC complexes target mRNAs that exhibit perfect sequence complementarity. Subsequently, siRNA-mediated gene silencing occurs by cleavage, release, and degradation of the target.

[0387] The siRNA duplex includes a sense strand that is homologous to the target mRNA and an antisense strand that is complementary to the target mRNA. It provides and uses single-stranded (ss)-siRNA (e.g., antisense strand RNA or antisense oligo) in terms of target RNA destruction efficiency. Nucleotides) have many advantages over In most cases, higher concentrations of ss-siRNA are required to achieve effective gene silencing efficacy for corresponding duplexes. [siRNA] [Double Helix Design and Sequence]

[0388] Several specifications for designing siRNA have been proposed in the art. These specifications generally recommend the generation of a 19-nucleotide duplex region, a symmetric 2-3 nucleotide 3' overhang, a 5'-phosphate group, and a 3'-hydroxyl group that targets the region in the gene to be silenced. Other rules that may govern siRNA sequence preference include, but are not limited to: (i) the presence of A / U at the 5' end of the antisense strand; (ii) the presence of G / C at the 5' end of the sense strand; (iii) the presence of G / C at the 5' end of the sense strand; There are at least five A / U residues in the 5' terminal third of the antisense strand; and (iv) there is no GC extension longer than 9 nucleotides. Based on such considerations, along with the specific sequence of the target gene, it is easy to design efficient siRNA constructs necessary to suppress the expression of the target gene in mammals.

[0389] In some embodiments, siRNA constructs (eg, siRNA duplexes or encoded dsRNA) are designed to target specific genes. Such siRNA constructs can specifically inhibit gene expression and protein production. In some aspects, siRNA constructs are designed and used to selectively "knock out" genetic variants in cells, i.e., identify mutated transcripts in patients that may be causative of various diseases and / or disorders. . In some aspects, siRNA constructs are designed and used to selectively "attenuate" variants of a gene in a cell. In other aspects, siRNA constructs can inhibit or suppress both wild-type and mutant forms of a gene.

[0390] In some embodiments, the siRNA sequence includes a sense strand and a complementary antisense strand, where the two strands hybridize together to form a double helix structure. The antisense strand has sufficient complementarity to the target mRNA sequence to direct target-specific RNAi, that is, the siRNA molecule has a sequence sufficient to trigger the destruction of the target mRNA by the RNAi mechanism or process.

[0391] In some embodiments, the siRNA sequence includes a sense strand and a complementary antisense strand, wherein the two strands hybridize together to form a double helix structure, and wherein the initiation site of hybridization to the mRNA is between nucleotides 100 and 100 on the mRNA sequence. Between 10,000. As a non-limiting example, the starting site can be at nucleotides 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500 on the mRNA sequence ,500-550,550-600,600-650,650-700,700-70,750-800,800-850,850-900,900-950,950-1000,1000-1050,1050-1100,1100 -1150, 1150-1200, 1200-1250, 1250-1300, 1300-1350, 1350-1400, 1400-1450, 1450-1500, 1500-1550, 1550-1600, 1600-1650, 1650-1700, 1700-1 750 ,1750-1800,1800-1850,1850-1900,1900-1950,1950-2000,2000-2050,2050-2100,2100-2150,2150-2200,2200-2250,2250-2300,2300-2350, 2350 -2400, 2400-2450, 2450-2500, 2500-2550, 2550-2600, 2600-2650, 2650-2700, 2700-2750, 2750-2800, 2800-2850, 2850-2900, 2900-2950, ​​2950-3 000 , 3000-3050, 3050-3100, 3100-3150, 3150-3200, 3200-3250, 3250-3300, 3300-3350, 3350-3400, 3400-3450, 3450-3500, 3500-3550, 3550-3600, 3600 -3650, 3650-3700, 3700-3750, 3750-3800, 3800-3850, 3850-3900, 3900-3950, 3950-4000, 4000-4050, 4050-4100, 4100-4150, 4150-4200, 4200-4 250 , 4250-4300, 4300-4350, 4350-4400, 4400-4450, 4450-4500, 4500-4550, 4550-4600, 4600-4650, 4650-4700, 4700-4750, 4750-4800, 4800-4850, 4850 -4900, 4900-4950, 4950-5000, 5000-5050, 5050-5100, 5100-5150, 5150-5200, 5200-5250, 5250-5300, 5300-5350, 5350-5400, 5400-5450, 5450-5 500 ,5500-5550,5550-5600,5600-5650,5650-5700,5700-5750,5750-5800,5800-5850,5850-5900,5900-5950,5950-6000,6000-6050,6050-6100, 6100 -6150, 6150-6200, 6200-6250, 6250-6300, 6300-6350, 6350-6400, 6400-6450, 6450-6500, 6500-6550, 6550-6600, 6600-6650, 6650-6700, 6700-6 750 , 6750-6800, 6800-6850, 6850-6900, 6900-6950, 6950-7000, 7000-7050, 7050-7100, 7100-7150, 7150-7200, 7200-7250, 7250-7300, 7300-7350, 7350 -7400, 7400-7450, 7450-7500, 7500-7550, 7550-7600, 7600-7650, 7650-7700, 7700-7750, 7750-7800, 7800-7850, 7850-7900, 7900-7950, 7950-8 000 , 8000-8050, 8050-8100, 8100-8150, 8150-8200, 8200-8250, 8250-8300, 8300-8350, 8350-8400, 8400-8450, 8450-8500, 8500-8550, 8550-8600, 8600 -8650, 8650-8700, 8700-8750, 8750-8800, 8800-8850, 8850-8900, 8900-8950, 8950-9000, 9000-9050, 9050-9100, 9100-9150, 9150-9200, 9200-9 250 ,9250-9300,9300-9350,9350-9400,9400-9450,9450-9500,9500-9550,9550-9600,9600-9650,9650-9700,9700-9750,9750-9800,9800-9850, 9850 -9900, 9900-9950, 9950-10000.

[0392] In some embodiments, the antisense strand has 100% complementarity to the target mRNA sequence. Antisense strands can be complementary to any part of the target mRNA sequence.

[0393] In other embodiments, the antisense strand contains at least one mismatch with the target mRNA sequence. As a non-limiting example, the antisense strand and the target mRNA sequence have at least 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or at least 20-30%, 20- 40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-99%, 30-40%, 30-50%, 30- 60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-99%, 40-50%, 40-60%, 40-70%, 40-80%, 40- 90%, 40-95%, 40-99%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 60-70%, 60- 80%, 60-90%, 60-95%, 60-99%, 70-80%, 70-90%, 70-95%, 70-99%, 80-90%, 80-95%, 80- 99%, 90-95%, 90-99% or 95-99% complementarity.

[0394] In some embodiments, the siRNA sequence is about 10-50 or more nucleotides in length, that is, each strand contains 10-50 nucleotides (or nucleotide analogs). Preferably, the length of the siRNA sequence in each strand is about 15-30, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, one of which is fully complementary to the target region. In some embodiments, the siRNA sequence is about 19 to 25, 19 to 24, or 19 to 21 nucleotides in length.

[0395] In some embodiments, the siRNA sequence can be a synthetic RNA double helix containing about 19 nucleotides to about 25 nucleotides and two overhanging nucleotides at the 3' end. In some aspects, the siRNA construct can be an unmodified RNA molecule. In other aspects, siRNA constructs can contain at least one modified nucleotide, such as a base, sugar, or backbone modification.

[0396] In some embodiments, siRNA sequences can be encoded in plasmid vectors, viral vectors, or other nucleic acid expression vectors for delivery to cells. DNA expression plasmids can be used to stably express siRNA double helices or dsRNA in cells and achieve long-term suppression of target gene expression. In one form, the sense and antisense strands of the siRNA duplex are often connected by short spacer sequences, resulting in the expression of a stem-loop structure called short hairpin RNA (shRNA). The hairpin is recognized and cleaved by Dicer, resulting in the mature siRNA construct.

[0397] In some embodiments, the sense and antisense strands of the siRNA duplex can be connected by a short spacer sequence, which can optionally be linked to additional flanking sequences, resulting in what is known as a primary microRNA (pri). - Representation of the flanking arm-stem-loop structure of miRNA). pri-miRNA can be recognized and cleaved by Drosha and Dicer, thus producing mature siRNA constructs.

[0398] In some embodiments, the siRNA duplex or encoded dsRNA inhibits (or degrades) the target mRNA. Therefore, the siRNA duplex or encoded dsRNA can be used to substantially inhibit gene expression in a cell. In some aspects, inhibition of gene expression refers to inhibition of at least about 20%, preferably at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100% , or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30- 40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40- 70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50- 100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80- 90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%. Therefore, the protein product of the targeted gene can be inhibited by at least about 20%, preferably at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, Or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40 %, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70 %, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100 %, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90 %, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%.

[0399] In some embodiments, the siRNA construct includes a miRNA seed match for the target located in the leader strand. In another embodiment, the siRNA construct contains a miRNA seed match for the target located in the follower strand. In yet another embodiment, the siRNA duplex or encoded dsRNA targeting a gene does not contain a seed match for the target located in the leader or follower strand.

[0400] In some embodiments, the siRNA duplex targeting the gene or the encoded dsRNA may have few significant full-length off-targets to the guide strand. In another embodiment, the siRNA duplex targeting the gene or the encoded dsRNA may have few significant full-length off-target effects on the follower strand. The siRNA double helix or encoded dsRNA targeting the gene can have less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 1-5%, 2-6%, 3-7%, 4- 8%, 5-9%, 5-10%, 6-10%, 5-15%, 5-20%, 5-25% 5-30%, 10-20%, 10-30%, 10-40 %, 10-50%, 15-30%, 15-40%, 15-45%, 20-40%, 20-50%, 25-50%, 30-40%, 30-50%, 35-50 %, 40-50%, 45-50% of full-length off-target effects. In another embodiment, the siRNA duplex targeting the gene or the encoded dsRNA may have almost no significant full-length off-target for the leader or follower strand. The siRNA double helix or encoded dsRNA targeting the gene can have less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 1-5%, 2-6%, 3-7% , 4-8%, 5-9%, 5-10%, 6-10%, 5-15%, 5-20%, 5-25%, 5-30%, 10-20%, 10-30% ,10-40%,10-50%,15-30%,15-40%,15-45%,20-40%,20-50%,25-50%,30-40%,30-50% , 35-50%, 40-50%, 45-50% full-length off-target effects.

[0401] In some embodiments, the siRNA duplex or encoded dsRNA targeting a gene may have high in vitro activity. In another embodiment, the siRNA construct may have low in vitro activity. In yet another embodiment, the siRNA duplex or dsRNA targeting the gene can have high leader strand activity and low follower strand activity in vitro.

[0402] In some embodiments, the siRNA construct has high in vitro leader activity and low in vitro follower activity. The target drawdown (KD) achieved by leading stocks can be at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5% or 100%. The target reduction achieved through guiding stocks can be 40-50%, 45-50%, 50-55%, 50-60%, 60-65%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 60-99%, 60-99.5%, 60-100%, 65-70%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 65-99%, 65-99.5%, 65-100%, 70-75%, 70-80%, 70-85%, 70-90%, 70-95%, 70-99%, 70-99.5%, 70-100%, 75-80%, 75-85%, 75-90%, 75-95%, 75-99%, 75-99.5%, 75-100%, 80-85%, 80-90%, 80-95%, 80-99%, 80-99.5%, 80-100%, 85-90%, 85-95%, 85-99%, 85-99.5%, 85-100%, 90-95%, 90-99%, 90-99.5%, 90-100%, 95-99%, 95-99.5%, 95-100%, 99-99.5%, 99-100% or 99.5-100%. As a non-limiting example, the target reduction (KD) achieved by leading stocks is greater than 70%. As a non-limiting example, the target reduction (KD) achieved by leading stocks is greater than 60%.

[0403] In some embodiments, the leader and follower (G:P) (also known as antonym and meaning) stock ratios are 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1;1, 2:10, 2:9, 2:8, 2:7, 2:6, 2:5, 2:4, 2: 3. 2:2, 2:1, 3:10, 3:9, 3:8, 3:7, 3:6, 3:5, 3:4, 3:3, 3:2, 3:1, 4:10, 4:9, 4:8, 4:7, 4:6, 4:5, 4:4, 4:3, 4:2, 4:1, 5:10, 5:9, 5: 8. 5:7, 5:6, 5:5, 5:4, 5:3, 5:2, 5:1, 6:10, 6:9, 6:8, 6:7, 6:6, 6:5, 6:4, 6:3, 6:2, 6:1, 7:10, 7:9, 7:8, 7:7, 7:6, 7:5, 7:4, 7: 3. 7:2, 7:1, 8:10, 8:9, 8:8, 8:7, 8:6, 8:5, 8:4, 8:3, 8:2, 8:1, 9:10, 9:9, 9:8, 9:7, 9:6, 9:5, 9:4, 9:3, 9:2, 9:1, 10:10, 10:9, 10: 8. 10:7, 10:6, 10:5, 10:4, 10:3, 10:2, 10:1, 1:99, 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85: 15. 90:10, 95:5 or 99:1 in vitro or in vivo. The leader-to-follower ratio refers to the ratio of leader strands to follower strands after intracellular processing of pri-microRNA. For example, a 80:20 lead:follower ratio would result in 8 lead strands for every 2 follower strands crafted by the precursor. As a non-limiting example, the ex vivo leader to follower ratio is 8:2. As a non-limiting example, the in vivo leader to follower ratio is 8:2. As a non-limiting example, the ex vivo leader to follower ratio is 9:1. As a non-limiting example, the in vivo leader to follower ratio is 9:1.

[0404] In some embodiments, the leader:follower (G:P) (also known as antonym:pride) ratio is greater than 1. In some embodiments, the leader:follower (G:P) (also known as antonym:pride) ratio is greater than 2. In some embodiments, the leader:follower (G:P) (also known as antonym:pride) ratio is greater than 5. In some embodiments, the leader:follower (G:P) (also known as antonym:pride) ratio is greater than 10. In some embodiments, the leader:follower (G:P) (also known as antonym:pride) ratio is greater than 20. In some embodiments, the leader:follower (G:P) (also known as antonym:sense) ratio is greater than 50. In some embodiments, the leader:follower (G:P) (also known as antonym:sense) ratio is at least 3:1. In some embodiments, the leader:follower (G:P) (also known as antonym:sense) ratio is at least 5:1. In some embodiments, the leader:follower (G:P) (also known as antonym:sense) ratio is at least 10:1. In some embodiments, the leader:follower (G:P) (also known as antonym:sense) ratio is at least 20:1. In some embodiments, the leader:follower (G:P) (also known as antonym:sense) ratio is at least 50:1.

[0405] In some embodiments, the follower:guidance (P:G) (also known as sense:antisense) ratios are 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1;1, 2:10, 2:9, 2:8, 2:7, 2:6, 2:5, 2:4, 2: 3. 2:2, 2:1, 3:10, 3:9, 3:8, 3:7, 3:6, 3:5, 3:4, 3:3, 3:2, 3:1, 4:10, 4:9, 4:8, 4:7, 4:6, 4:5, 4:4, 4:3, 4:2, 4:1, 5:10, 5:9, 5: 8. 5:7, 5:6, 5:5, 5:4, 5:3, 5:2, 5:1, 6:10, 6:9, 6:8, 6:7, 6:6, 6:5, 6:4, 6:3, 6:2, 6:1, 7:10, 7:9, 7:8, 7:7, 7:6, 7:5, 7:4, 7: 3. 7:2, 7:1, 8:10, 8:9, 8:8, 8:7, 8:6, 8:5, 8:4, 8:3, 8:2, 8:1, 9:10, 9:9, 9:8, 9:7, 9:6, 9:5, 9:4, 9:3, 9:2, 9:1, 10:10, 10:9, 10: 8. 10:7, 10:6, 10:5, 10:4, 10:3, 10:2, 10:1, 1:99, 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85: 15. 90:10, 95:5 or 99:1 in vitro or in vivo. The follower:leader ratio refers to the ratio of follower:leader after cutting off the leader. For example, a 80:20 Follower:Leader ratio would be crafted from Precursor with 8 Follower strands for every 2 Lead strands. As a non-limiting example, the in vitro follower:leader ratio is 80:20. As a non-limiting example, the in vivo follower:leader ratio is 80:20. As a non-limiting example, the in vitro follower:leader ratio is 8:2. As a non-limiting example, the in vivo follower:leader ratio is 8:2. As a non-limiting example, the in vitro follower:leader ratio is 9:1. As a non-limiting example, the in vivo follower:leader ratio is 9:1.

[0406] In some embodiments, the following followed:lead (P:G) (also known as sense:antisense) ratio is greater than 1. In some embodiments, the following followed:lead (P:G) (also known as sense:antisense) ratio is greater than 2. In some embodiments, the following followed:lead (P:G) (also known as sense:antisense) ratio is greater than 5. In some embodiments, the following-to-leader (P:G) (also known as sense:antisense) ratio is greater than 10. In some embodiments, the following-to-leader (P:G) (also known as sense:antisense) ratio is greater than 20. In some embodiments, the Follower:Leader (P:G) (also known as sense:antisense) ratio is greater than 50. In some embodiments, the follower:leader (P:G) (also known as sense:antisense) ratio is at least 3:1. In some embodiments, the follower:leader (P:G) (also known as sense:antisense) ratio is at least 5:1. In some embodiments, the follower:leader (P:G) (also known as sense:antense) ratio is at least 10:1. In some embodiments, the follower:leader (P:G) (also known as sense:antense) ratio is at least 20:1. In some embodiments, the follower:leader (P:G) (also known as sense:antisense) ratio is at least 50:1.

[0407] In some embodiments, when pri- or pre-microRNA but methods known in the art and described herein exhibit greater than a 2-fold leader to follower ratio, the follower-leader dual Spiral is considered valid. As a non-limiting example, when measured, pri-microRNA or pre-microRNA exhibits greater than 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times , 11 times, 12 times, 13 times, 14 times, 15 times or 2 to 5 times, 2 to 10 times, 2 to 15 times, 3 to 5 times, 3 to 10 times, 3 to 15 times, 4 to 5 times , 4 to 10 times, 4 to 15 times, 5 to 10 times, 5 to 15 times, 6 to 10 times, 6 to 15 times, 7 to 10 times, 7 to 15 times, 8 to 10 times, 8 to 15 times , 9 to 10 times, 9 to 15 times, 10 to 15 times, 11 to 15 times, 12 to 15 times, 13 to 15 times or 14 to 15 times the leading stock: following stock ratio.

[0408] In some embodiments, the vector genome encoding a dsRNA comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more than 99% of the full length of the construct % sequence. As a non-limiting example, the vector genome contains a sequence that is at least 80% of the full-length sequence of the construct.

[0409] In some embodiments, siRNA constructs can be used to silence wild-type or mutant genes by targeting at least one exon on the sequence. [siRNA] [modification]

[0410] In some embodiments, when not delivered as a precursor or DNA, the siRNA construct can be chemically modified to modulate some characteristics of the RNA molecule, such as, but not limited to, improving the stability of the siRNA in vivo. Chemically modified siRNA constructs can be used in human therapeutic applications and can be modified without compromising the RNAi activity of the siRNA construct. As a non-limiting example, the siRNA construct is modified at both the 3' and 5' ends of the sense and antisense strands.

[0411] In some embodiments, modified nucleotides may be only on the sense strand.

[0412] In some embodiments, modified nucleotides may be only on the antisense strand.

[0413] In some embodiments, modified nucleotides can be in both sense and antisense.

[0414] In some embodiments, chemically modified nucleotides do not affect the ability of the antisense strand to pair with the target mRNA sequence. [tiny] [RNA (miR)] [skeleton]

[0415] In some embodiments, siRNA constructs can be encoded in polynucleotide sequences that also include microRNA (miR) backbone constructs. As used herein, a "microRNA (miR) backbone construct" is a framework or starting molecule that forms the sequence or structural basis for designing or making subsequent molecules.

[0416] In some embodiments, a miR backbone construct includes at least one 5' flanking region. As a non-limiting example, the 5' flanking region may comprise a 5' flanking sequence that may be of any length and may be fully or partially derived from a wild-type microRNA sequence or be a completely artificial sequence.

[0417] In some embodiments, a miR backbone construct includes at least one 3' flanking region. As a non-limiting example, the 3' flanking region may comprise a 3' flanking sequence that may be of any length and may be fully or partially derived from a wild-type microRNA sequence or be a completely artificial sequence.

[0418] In some embodiments, a miR scaffold construct includes at least one ring motif region. As a non-limiting example, a loop motif region can include sequences that can be of any length.

[0419] In some embodiments, a miR backbone construct includes a 5' flanking region, a ring motif region, and / or a 3' flanking region.

[0420] In some embodiments, at least one payload (eg, siRNA, miRNA, or other RNAi agent described herein) can be encoded by a polynucleotide that can also include at least one miR scaffold construct. The miR backbone construct may comprise 5' flanking sequences that may be of any length and may be derived entirely or partially from wild-type microRNA sequences or entirely artificial. The 3' flanking sequence may be a mirror image of the 5' flanking sequence and / or the 3' flanking sequence in size and origin. There may not be any flanking sequences. The 3' flanking sequence optionally contains one or more CNNC motifs, where "N" represents any nucleotide.

[0421] In some embodiments, the 5' arm of the stem-loop structure of a polynucleotide comprising or encoding a miR framework construct includes a sequence encoding a sense sequence.

[0422] In some embodiments, the 3' arm of the stem loop of the polynucleotide comprising or encoding the miR scaffold construct includes a sequence encoding an antisense sequence. In some cases, the antisense sequence contains a "G" nucleotide at the 5'-most end.

[0423] In some embodiments, the sense sequence can reside on the 3' arm of the stem-loop structure of the polynucleotide comprising or encoding the miR framework construct, while the antisense sequence resides on the 5' arm thereof.

[0424] In some embodiments, the sense and antisense sequences may be completely complementary throughout most of their length. In other embodiments, the sense sequence and the antisense sequence can be present in at least 70% of the length of each strand, independently at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the length of each strand. %, 80%, 90%, 95% or 99% complementarity.

[0425] Neither the sense sequence identity nor the antisense sequence homology need to be 100% complementary to the target sequence.

[0426] In some embodiments, what separates the sense and antisense sequences of the stem-loop structure of the polynucleotide is a loop sequence (also known as a loop motif, linker, or linker motif). The loop sequence can be of any length: between 4-30 nucleotides, between 4-20 nucleotides, between 4-15 nucleotides, between 5-15 nucleotides , between 6-12 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides and / or 15 nucleotides.

[0427] In some embodiments, the loop sequence includes a nucleic acid sequence encoding at least one UGUG motif. In some embodiments, the nucleic acid sequence encoding the UGUG motif is located at the 5' end of the loop sequence.

[0428] In some embodiments, spacer regions may be present in the polynucleotide to separate one or more modules (e.g., 5' flanking region, loop motif region, 3' flanking region, sense sequence, antisense sequence sequences) are separated from each other. There may be one or more such spaced regions.

[0429] In some embodiments, a spacer having between 8 and 20, that is, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides may Present between the sense sequence and the flanking region sequence.

[0430] In some embodiments, the spacer region is 13 nucleotides in length and is located between the 5' end of the sense sequence and the 3' end of the flanking sequence. In some embodiments, the spacer is of sufficient length to form approximately one helical turn of the sequence.

[0431] In some embodiments, a spacer region having between 8 and 20, that is, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides may Present between the antisense sequence and the flanking sequence.

[0432] In some embodiments, the spacer sequence is between 10-13, that is, 10, 11, 12, or 13 nucleotides, and is located between the 3' end of the antisense sequence and the 5' end of the flanking sequence. In some embodiments, the spacer is of sufficient length to form approximately one helical turn of the sequence.

[0433] In some embodiments, the polynucleotide includes a 5' flanking sequence, a 5' arm, a loop motif, a 3' arm, and a 3' flanking sequence in the 5' to 3' direction. As a non-limiting example, the 5' arm can comprise a sense sequence and the 3' arm an antisense sequence. In another non-limiting example, the 5' arm contains antisense sequences and the 3' arm contains sense sequences.

[0434] In some embodiments, the 5' arm, payload (e.g., sense sequence and / or antisense sequence), loop motif, and / or 3' arm sequence may be altered (e.g., substituting one or more nucleotides, adding core nucleotides and / or missing nucleotides). This change can cause beneficial changes in the function of the construct (e.g., increased attenuation of gene expression of the target sequence, reduced degradation of the construct, reduced off-target effects, increased efficiency of the payload, and reduced degradation of the payload).

[0435] In some embodiments, the miR backbone constructs of polynucleotides are aligned so that the resection rate of the leading strand is greater than the resection rate of the follower strand. The resection rate of the leading stock or the following stock can be independently 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more than 99%. As a non-limiting example, the resection rate of the guide strand is at least 80%. As another non-limiting example, the resection rate of the guide strand is at least 90%.

[0436] In some embodiments, the resection rate of the leading strand is greater than the resection rate of the following strand. In a pattern, the removal rate of the leading stock can be at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40 greater than that of the following stock. %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more than 99%.

[0437] In some embodiments, the guide strand has a resection efficiency of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more than 99%. As a non-limiting example, the resection efficiency of the guide strand is greater than 80%.

[0438] In some embodiments, resection efficiency of the leading femur is greater than resection of the follower femur from the miR scaffold construct. Resection of the leading strand can be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times more efficient than resection of the following strand from the miR skeleton construct.

[0439] In some embodiments, a miR scaffold construct includes a bifunctional targeting polynucleotide. As used herein, a "bifunctional targeting" polynucleotide is a polynucleotide in which the leader and follower strands attenuate the same target or in which the leader and follower strands attenuate different targets.

[0440] In some embodiments, the miR scaffold constructs of polynucleotides described herein can include a 5' flanking region, a loop motif region, and a 3' flanking region.

[0441] In some embodiments, polynucleotides are designed using at least one of the following properties: loop variants, seed mismatch / bump / wobble variants, stem mismatches, loop variants, and vassal stem mismatches Matching variants, seed mismatch and basal stem mismatch variants, stem mismatch and basal stem mismatch variants, seed wiggle and basal stem wiggle variants or stem sequence variants.

[0442] In some embodiments, the miR backbone construct can be a native pri-miRNA backbone.

[0443] In some embodiments, the selection of a miR backbone construct is determined by comparing polynucleotides in pri-miRNA.

[0444] In some embodiments, the selection of a miR backbone construct is determined by comparing polynucleotides in natural pri-miRNA and synthetic pri-miRNA. [Transfer] [RNA] [(tRNA)]

[0445] Transfer RNA (tRNA) is the RNA molecule that translates mRNA into protein. tRNA includes a cloverleaf structure including a 3' acceptor site, a 5' terminal phosphate, a D arm, a T arm and an anticodon arm. The main purpose of tRNA is to carry the amino acid on its 3' acceptor site to the Ribosomal complexes. Once an amino acid binds to a tRNA, the tRNA is considered an amino acid-tRNA. The type of amino acid on the tRNA depends on the codon of the mRNA. The anticodon arm of tRNA is the anticodon site, which is complementary to the mRNA codon and determines which amino acid is carried. tRNA is also known to have a role in regulating apoptosis by acting as a cytochrome c scavenger.

[0446] In some embodiments, the initial construct and / or the reference construct includes or encodes tRNA. [Ribosome] [RNA] [(rRNA)]

[0447] Ribosomal RNA (rRNA) is the RNA that forms ribosomes. The ribose system is necessary for protein synthesis and contains large ribosome subunits and small ribosome subunits. In prokaryotes, the small 30S and large 50S ribosome subunits constitute the 70S ribosome. In eukaryotes, the 40S and 60S subunits form the 80S ribosome. To bind aminoacyl-tRNA and link amino acids together to create polypeptides, ribosomes contain three types of sites: exit site (E), peptidyl site (P), and acceptor site (A) .

[0448] In some embodiments, the initial construct and / or the reference construct comprises or encodes rRNA. [tiny] [RNA (miRNA)]

[0449] MicroRNA (or miRNA) is a 19-25 nucleotide long non-coding RNA that binds to the 3'UTR of nucleic acid molecules and downregulates gene expression by reducing the stability of nucleic acid molecules or inhibiting translation. The initial construct and / or the reference construct may include one or more microRNA target sequences, microRNA sequences or microRNA seeds.

[0450] The microRNA sequence contains the "seed" region, that is, the sequence in the region of positions 2-8 of the mature microRNA, which has perfect Watson-Crick complementarity with the miRNA target sequence. The microRNA seed may contain positions 2-8 or 2-7 of the mature microRNA. In some embodiments, a microRNA seed can comprise 7 nucleotides (e.g., nucleotides 2-8 of a mature microRNA), where the seed complementary site corresponding to the miRNA target consists of an adenine opposite position 1 of the microRNA. (A) Side connection. In some embodiments, a microRNA seed can comprise 6 nucleotides (e.g., nucleotides 2-7 of a mature microRNA), where the seed complementary site in the corresponding miRNA target consists of an adenine opposite position 1 of the microRNA. (A) Side connection. The bases of microRNA seeds are completely complementary to the target sequence. By engineering microRNA target sequences into the 3' UTR of the mRNA, we can target molecules for degradation or reduced translation, subject to the constraints available for the microRNA in question. This process will reduce the risk of off-target effects when delivering nucleic acid molecules.

[0451] As used herein, the term "microRNA site" refers to a microRNA target site or a microRNA recognition site, or any nucleotide sequence that binds or associates with a microRNA. It is understood that "binding" may follow traditional Watson-Crick hybridization rules, or may reflect any stable association of a microRNA with a target sequence at or near the microRNA site.

[0452] Non-limiting examples of tissues where microRNAs are known to regulate mRNA, and thus protein expression, include, but are not limited to, liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17 -92,miR-126), bone marrow cells (miR-142-3p,miR-142-5p,miR-16,miR-21,miR-223,miR-24,miR-27), adipose tissue (let-7 ,miR-30c), heart (miR-ld,miR-149), kidney (miR-192,miR-194,miR-204) and lung epithelial cells (let-7,miR-133,miR-126). MicroRNA can also regulate complex biological processes, such as angiogenesis (miR-132).

[0453] For example, if the nucleic acid molecule is mRNA and is not intended to be delivered to the liver but ends up there, then miR-122 (a microRNA that is abundant in the liver) can pass through one or more of the target sites of miR-122. Suppresses the expression of the gene of interest when engineered into the 3' UTR of the mRNA. The introduction of one or more binding sites for different microRNAs can be engineered to further reduce the longevity, stability, and protein translation of the mRNA.

[0454] Conversely, microRNA binding sites can be engineered outside (i.e., removed from) the sequence in which they naturally occur in order to increase protein expression in specific tissues. For example, the miR-122 binding site can be removed to improve protein expression in the liver. Modulation of expression in multiple tissues can be achieved through the introduction or removal of one or several microRNA binding sites. [long uncoded] [RNA (lncRNA)]

[0455] Long non-coding RNA (lncRNA) is a regulatory RNA molecule that does not encode proteins but affects a large number of biological processes. lncRNA designations are generally restricted to non-coding transcripts longer than approximately 200 nucleotides. The length designation distinguishes lncRNAs from small regulatory RNAs such as short interfering RNAs (siRNAs) and microRNAs (miRNAs). In vertebrates, the number of lncRNA species is believed to greatly exceed the number of protein-coding species. It is also believed that lncRNA drives the biological complexity observed in vertebrates compared to invertebrates. Evidence of this complexity can be found in many cellular compartments of vertebrate organisms, such as the T lymphocyte compartment of the adaptive immune system. Differences in expression and function of lncRNA may be the main cause of human diseases.

[0456] In some embodiments, the initial construct and / or the baseline construct includes lncRNA. [RNA] [modification]

[0457] In some aspects, the initial construct or baseline construct may contain one or more modified nucleotides, such as, but not limited to, sugar-modified nucleotides, nucleobase modifications, and / or backbone modifications. In some aspects, the initial construct or baseline construct may contain combined modifications, such as combined nucleobase and backbone modifications.

[0458] In some embodiments, the modified nucleotides may be sugar-modified nucleotides. Sugar-modified nucleotides include, but are not limited to, 2'-fluoro, 2'-amino and 2'-thio modified ribonucleotides, such as 2'-fluoro modified ribonucleotides. Modified nucleotides can be modified on the sugar moiety, as well as nucleotides having sugars or non-ribosyl analogs thereof. For example, the sugar moiety may be or be based on mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose and other sugars, heterocycles or carbocycles.

[0459] In some embodiments, the modified nucleotides may be nucleobase modified nucleotides.

[0460] In some embodiments, the modified nucleotide can be a backbone modified nucleotide. In some embodiments, the initial construct or baseline construct may further include other modifications on the backbone. As used herein, commonly used "backbone" refers to the repeating alternating phosphate sugar sequence in a DNA or RNA molecule. The deoxynucleoside / ribose sugar is joined to the phosphate group at the 3'-hydroxyl and 5'-hydroxyl groups via ester bonds (also known as "phosphodiester" bonds / linkers (PO bonds)). The PO backbone can be modified to a "phosphorothioate backbone (PS linkage)". In some cases, the natural phosphodiester bond can be replaced by an amide bond, but the four atoms between the two sugar units are maintained. Such amide modification can facilitate solid-phase synthesis of oligonucleotides and improve the thermodynamic stability of the double helix formed with the complementary sequence of siRNA.

[0461] Modified bases refer to nucleotide bases such as, but not limited to, adenine, guanine, cytosine, thymine, uracil, xanthine, inosine, and Q nucleosides, which have been modified by substitution or addition of a or multiple atoms or groups modified. Some examples of modifications on nucleobase moieties include, but are not limited to, alkylated, halogenated, thiolated, aminated, amidated or acetylated bases, independently or in combination. More specific examples include, for example, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N,-dimethyladenine, 2-propynyl Adenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and those with Modified other nucleotides, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine Adenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine Glycosides, 5-methoxyuridine, deazonucleotides (such as 7-deazo-adenosine, 6-azoyuridine, 6-azocytidine, 6-azothymidine), 5-Methyl-2-thiouridine, other thiobases (such as 2-thiouridine and 4-thiouridine and 2-thiocytidine), dihydrouridine, pseudouridine, Q nucleosides, ancient purines, naphthyl and substituted naphthyl, any O-alkylated purines and pyrimidines and N-alkylated purines and pyrimidines (such as N6-methyladenosine, 5-methylcarbonylmethyl uridine, uridine 5-oxyacetate, pyridin-4-one, pyridin-2-one), phenyl and modified phenyl (such as aminophenol or 2,4,6-trimethoxybenzene) , modified cytosine (which acts as a G-shaped clamp nucleotide), 8-substituted adenine and guanine, 5-substituted uracil and thymine, azapyrimidine, carboxyhydroxyalkyl nucleotide, carboxyl Alkylamine nucleotides and alkylcarbonylalkylated nucleotides.

[0462] Initial constructs and / or reference constructs that may include one or more substitutions, insertions and / or additions, deletions and covalent modifications relative to the reference sequence (in particular, the parent RNA) are included within the scope of the invention. .

[0463] In some embodiments, the initial construct and / or the baseline construct includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, polyA sequence, methylation, chelation, phosphorylation , methylation, acetylation of lysine and arginine residues and nitrosylation of thiol and tyrosine residues, etc.). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of over a hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197). In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the initial construct and / or the baseline construct includes at least one nucleoside selected from the group consisting of: pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio- 5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl methyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinemethyluridine, 1-taurinemethyl methyl-pseudouridine, 5-taurinemethyl-2-thio-uridine, 1-taurinemethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl -pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-desazo-pseudouridine, 2-thio Base-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2- Methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine and 4-methoxy-2-thio-pseudouridine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of: 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-methanoylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thiocytidine Cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1- Methyl-1-desa-pseudocytidine, 1-methyl-1-desa-pseudocytidine, zebularine, 5-aza-zebularine, 5-methyl -Zebrane, 5-aza-2-thio-Zebrane, 2-thio-Zebrane, 2-methoxy-cytidine, 2-methoxy-5-methyl - Cytidine, 4-methoxy-pseudoisocytidine and 4-methoxy-1-methyl-pseudoisocytidine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of: 2-aminopurine, 2,6-diaminopurine, 7-desaza-adenine, 7-desaza-8 -Aza-adenine, 7-desa-2-aminopurine, 7-desa-8-aza-2-aminopurine, 7-desa-2,6-diaminopurine, 7- Deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-prenyladenosine, N6-(cis-hydroxyisopentene methyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylamine methyladenosine, N6-threonylamine methyladenosine Glycoside, 2-methylthio-N6-threonylamine methyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine and 2-methyladenosine Oxy-adenine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of: inosine, 1-methyl-inosine, wyosine, wybutosine, 7-desine Nitrogen-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-guanosine -Aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine , N2-methylguanosine, N2,N2-dimethylguanosine, 8-side oxy-guanosine, 7-methyl-8-side oxy-guanosine, 1-methyl-6-sulfanyl -Guanosine, N2-methyl-6-thio-guanosine and N2,N2-dimethyl-6-thio-guanosine.

[0464] The initial construct and / or the baseline construct may include any suitable modifications such as sugars, nucleobases, or internucleoside linkages (eg, connecting phosphate / phosphodiester linkages / phosphodiester backbones). One or more atoms of the pyrimidine nucleobase may be optionally substituted with an amine group, an optionally substituted thiol, an optionally substituted alkyl group (such as methyl or ethyl), or a halo group (such as chlorine or ethyl). Fluorine) replacement or substitution. In certain embodiments, modifications (eg, one or more modifications) are present in each of the sugar and internucleoside linkage. The modification may be ribonucleic acid (RNA) modified into deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA) or a mixture thereof. This article describes additional modifications.

[0465] In some embodiments, the initial construct and / or the baseline construct includes at least one N(6)methyladenosine (m6A) modification that increases translation efficiency. In some embodiments, N(6)methyladenosine (m6A) modification can reduce the immunogenicity of the initial construct and / or the baseline construct.

[0466] In some embodiments, modifications may include chemical or cell-induced modifications. For example, some non-limiting examples of intracellular RNA modifications are provided by Lewis and Pan in "RNA modifications and structures cooperate to guide RNA-protein interactions", Nat. Reviews Mol. Cell Biol., 2017, 18:202-210 describe.

[0467] In some embodiments, chemical modification of RNA can enhance immune evasion. RNA can be synthesized and / or modified by methods well established in the art, such as those described in: "Current protocols in nucleic acid chemistry", Beaucage, S. L. et al. (eds.), John Wiley & Sons , Inc., New York, N.Y., USA, which is incorporated herein by reference. Modifications include, for example, end modifications, such as 5' end modifications (phosphorylation (single, double and triple), conjugation, reverse bonding), 3' end modifications (conjugation, DNA nucleotides, reverse bonding, etc.), base Base modification (e.g., base substitution via stabilizing base, unstable base, or base pairing with an expanded library of partners), removal of bases (abasic nucleotides), or conjugate bases. Modified ribonucleotide bases may also include 5-methylcytidine and pseudouridine. In some embodiments, base modifications can modulate RNA performance, immune response, stability, subcellular localization, to name just a few functional roles. In some embodiments, modifications include diorthogonal nucleotides, such as non-natural bases. See, for example, Kimoto et al., Chem Commun (Camb), 2017, 53:12309, DOI: 10.1039 / c7cc06661a, which is incorporated herein by reference.

[0468] In some embodiments, sugar modifications (eg, at the 2' position or 4' position) or substitutions of one or more RNA sugars as well as backbone modifications may include modifications or substitutions of phosphodiester linkages. Specific examples of modifications include modified backbones or absence of natural internucleoside linkages, such as internucleoside modifications, including modification or replacement of phosphodiester linkages. RNAs with modified backbones particularly include those without phosphorus atoms in the backbone. For the purposes of this application, and as is sometimes mentioned in the art, modified RNAs that do not have phosphorus atoms in the internucleoside backbone may also be considered oligonucleotides. In specific embodiments, RNA will include ribonucleotides having a phosphorus atom in its internucleoside backbone.

[0469] Modified RNA backbones may include, for example, phosphorothioates; parachiral phosphorothioates; phosphorodithioates; phosphate triesters; aminoalkyl phosphate triesters; methyl and other alkyl phosphonates, Such as 3'-alkylenephosphonates and p-chiral phosphonates; phosphonites; aminophosphates such as 3'-aminoaminophosphates with normal 3'-5' linkage and amines Alkylamino phosphates, thioamido phosphates, thiocarbonyl alkyl phosphonates, thiocarbonyl alkyl triesters and borane phosphates, their 2'-5' linked analogs and reverse Polar amino phosphates in which pairs of adjacent nucleoside units are connected 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts and free acid forms. In some embodiments, RNA can be negatively or positively charged.

[0470] Modified nucleotides can be modified at internucleoside linkages (eg, phosphate backbone). Herein, in the context of polynucleotide backbones, the phrases "phosphate ester" and "phosphodiester" are used interchangeably. The backbone phosphate group can be modified by replacing one or more oxygen atoms with different substituents. Additionally, modified nucleosides and nucleotides may include bulk replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, selenophosphate, boranophosphate, boranophosphate ester, hydrophosphonate, aminophosphate, Diaminophosphates, alkyl or aryl phosphonates and phosphate triesters. Both non-attached oxygens of phosphorodithioates are replaced by sulfur. Phosphate linkers can also be modified by replacing the connecting oxygen with nitrogen (bridged amine phosphate), sulfur (bridged phosphorothioate), and carbon (bridged methylene-phosphonate).

[0471] Alpha-thio-substituted phosphate moieties are provided to impart stability to RNA and DNA polymers via these non-natural phosphorothioate backbone bonds. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently have a longer half-life in the cellular environment. Phosphorothioates linked to RNA are expected to reduce innate immune responses through weaker binding / activation of cellular innate immune molecules.

[0472] In specific embodiments, modified nucleosides include alpha-thio-nucleosides (e.g., 5'-O-(1-phosphorothioate)-adenosine, 5'-O-(1-phosphorothioate) )-cytidine (a-thio-cytidine), 5'-O-(1-phosphorothioate)-guanosine, 5'-O-(1-phosphorothioate)-uridine or 5' -O-(1-phosphorothioate)-pseudouridine).

[0473] Other internucleoside linkages that may be employed in accordance with the present invention are described herein, including internucleoside linkages that do not contain phosphorus atoms.

[0474] In some embodiments, the RNA may include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides can be incorporated into RNA, such as bifunctional modifications. Cytotoxic nucleosides may include, but are not limited to, vidarabine, 5-azacytidine, 4'-thio-cytarabine, cyclopentenylcytosine, cladribine, clofarabine ( clofarabine), cytarabine, cytosine arabinosine, l-(2-C-cyano-2-deoxy-β-D-arabinose-pentofuranosyl)-cytosine, decetaxel decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, combination of tegafur and uracil, tegafur ((RS)-5-fluoro-l-(tetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione), troxacitabine, tezacitabine, 2'-Deoxy-2'-methylenecytosine (DMDC) and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-docanoyl-1-β-D-arabinofuranosine, N4-octadecyl-1-β-D-arabinofuranosine Cytosine, N4-palmitoyl-1-(2-C-cyano-2-deoxy-β-D-arabinose-pentofuranosyl)cytosine and P-4055 (cytarabine 5' - Edelaid).

[0475] In some embodiments, the RNA sequence includes or includes natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, myosine Glycoside, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propyn...

Claims

1. A compound of formula IC or a pharmaceutically acceptable salt thereof, IC, wherein: A is selected from the group consisting of free radicals and alkyl groups; R1a is -L1-R1; L1 is a C2-C6 alkyl group; R1 is selected from the group consisting of -OH, , , , , , , and ; R2a, R2b and R2c are independently selected from the group consisting of hydrogen and C1-C6 alkyl groups; R3a, R3b and R3c are independently selected from the group consisting of hydrogen and C1-C6 alkyl groups; R4a, R4b and R4c are independently selected from the group consisting of hydrogen and C1-C6 alkyl groups; R5a, R5b and R5c are independently selected from the group consisting of hydrogen and C1-C6 alkyl groups; R6a, R6b and R6c are independently selected from the group consisting of hydrogen and C1-C6 alkyl groups; or R6a and R6b are bonded to the nitrogen atom to form a 4 to 8-membered heterocycle; and R6c is selected from the group consisting of hydrogen and C1-C6 alkyl groups; R7a, R7b, and R7c are independently selected from the group consisting of hydrogen and C1-C6 alkyl groups; or R7a and R7b are bonded to the nitrogen atom to which they are attached to form a 4- to 8-membered heterocycle; and R7c is selected from the group consisting of hydrogen and C1-C6 alkyl groups; R' is hydrogen; R8a is -L2-R8; L2 is a C2-C6 alkyl group; R8 is selected from the group consisting of -NR9aR9b, ...; R9a and R9b are independently selected from the group consisting of hydrogen and C1-C6 alkyl groups; or R9a and R9b are bonded to the nitrogen atom to form a 4- to 8-membered heterocycle; Q1 is a C1-C20 alkyl group; W1 is selected from the group consisting of: -C(=O)O-, -OC(=O)-, -C(=O)N(R12a)-, -N(R12a)C(=O)-, -OC(=O)N(R12a)-, -N(R12a)C(=O)O-, and -OC(=O)O-; R12a is selected from the group consisting of hydrogen and C1-C6 alkyl groups; X1 is a substituted branched C1-C15 alkyl group, or X1 is a bond; Y1 is selected from the group consisting of -(CH2)m-, -O-, -S-, and -SS-; m is 0, 1, 2, 3, 4, 5, or 6; Z1 is a substituted C5-C12 bridged cycloalkyl group, as appropriate; R10 is selected from the group consisting of hydrogen, C1-C20 alkyl groups, and C2-C20 alkenyl groups; Q2 is a C1-C20 alkyl group; W2 is selected from the group consisting of -C(=O)O-, -OC(=O)-, -C(=O)N(R12b)-, -OC(=O)N(R12b)- and -OC(=O)O-; R12b is selected from the group consisting of hydrogen and C1-C6 alkyl groups; X2 is a C1-C15 branched alkyl group that is substituted as appropriate; Y2 is -(CH2)n-; n is 0, 1, 2, 3, 4, 5 or 6; Z2 is -(CH2)p-; p is 0 or 1; and R11 is a C1-C20 branched alkyl group, wherein one or more methylene bonds of R11 are substituted as appropriate and independently by groups selected from -O- and -CH=CH-.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula II: II, or a pharmaceutically acceptable salt thereof.

3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula III: III, or a pharmaceutically acceptable salt thereof.

4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein W1 is -C(=O)O- or -OC(=O)-, and wherein W2 is -C(=O)O- or -OC(=O)-.

5. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein Q1 is selected from the group consisting of: -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, -CH2(CH2)4CH2- and -CH2(CH2)5CH2-, and Q2 is selected from the group consisting of: -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, -CH2(CH2)4CH2- and -CH2(CH2)5CH2-.

6. The compound of claim 1 or its pharmaceutically acceptable salt, wherein X1 is a bond.

7. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein X2 is, where appropriate, a substituted C1-C15 branched alkyl group.

8. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein -Q1-W1-X1-Y1-Z1-R10 is selected from the group consisting of:

9. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein -Q2-W2-X2-Y2-Z2-R11 is selected from the group consisting of:

10. A compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula XII'': XII'' or a pharmaceutically acceptable salt thereof, wherein q1 is 0, 1, 2 or 3; q2 is 0, 1, 2 or 3; r2 is 0, 1 or 2; and s2 is 0, 1, 2, 3, 4, 5 or 6.

11. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula XIII': XIII' or a pharmaceutically acceptable salt thereof, wherein q1 is 0, 1, 2 or 3; q2 is 0, 1, 2 or 3; r2 is 0, 1 or 2; and s2 is 0, 1, 2, 3, 4, 5 or 6.

12. The compound of claim 1 or its pharmaceutically acceptable salt, wherein Z1 is selected from the group consisting of: , , , , , , and.

13. The compound of claim 1 or its pharmaceutically acceptable salt, wherein Z1 is selected from the group consisting of: , , and; and R10 is hydrogen.

14. A compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula XVII: XVII or a pharmaceutically acceptable salt thereof, wherein q1 is 0, 1, 2 or 3; q2 is 0, 1, 2 or 3.

15. A compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula XIX: XIX or a pharmaceutically acceptable salt thereof, wherein q1 is 0, 1, 2 or 3; q2 is 0, 1, 2 or 3.

16. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein L1 is selected from the group consisting of -CH2CH2-, -CH2CH2CH2- and -CH2CH2CH2CH2-; or L2 is selected from the group consisting of -CH2CH2-, -CH2CH2CH2- and -CH2CH2CH2CH2-.

17. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R11 is a C1-C15 branched alkyl group.

18. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from: or a pharmaceutically acceptable salt thereof.

19. A lipid nanoparticle (LNP) comprising an ionizable lipid, wherein the ionizable lipid is a compound of any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof.

20. The LNP of claim 19 further comprises: (a) PEG-lipids; (b) structural lipids; and (c) non-ionizable lipids and / or zwitterionic lipids.

21. The LNP of claim 20, wherein the PEG-lipid is selected from the group consisting of: PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC and PEG-DSPE.

22. The LNP of claim 20, wherein the structural lipid is selected from the group consisting of: cholesterol, coprosterol, phytosterol, ergosterol, campesterol, stigmasterol, phytosterol, ursolic acid, and α-tocopherol.

23. The LNP of claim 20, wherein the non-ionizable lipid is a phospholipid selected from the group consisting of: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE ... 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DMPC) hocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (1-palmitoyl-2-oleyl-2 ... yl-2-oleoyl-sn-glycero-3-phosphocholine; POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine; 18:0 diether PC), 1-oleoyl-2-cholesterylhemisuc cinoyl-sn-glycero-3-phosphocholine; OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine;C16 Lyso PC), 1,2-dilinalinyl-sn-glycero-3-phosphocholine, 1,2-diarachidonicyl-sn-glycero-3-phosphocholine, 1,2-bis(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-diphytanoylsn-glycero-3-phosphoethanolamine (ME 16.0) PE), 1,2-distearyl-sn-glycero-3-phosphate ethanolamine, 1,2-dilinoleyl-sn-glycero-3-phosphate ethanolamine, 1,2-dilinoleyl-sn-glycero-3-phosphate ethanolamine, 1,2-diarachidonicyl-sn-glycero-3-phosphate ethanolamine, 1,2-bis(docosahexaenoyl)-sn-glycero-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphate sodium salt (1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt) Sodium (S)-2-ammonium-3-((((R)-2-(oleyloxy)-3-(stearyloxy)propoxy)oxophosphatidyl)oxy)propionate (L-α-phosphatidylinosine; brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylcholine (DMPC) Phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (1,2-dioleoyl-sn-glycero-3-(phospho-L-serine; DOPS), acell-fusogenic phospholipid (DPhPE), dipalmitoylphosphatidylethanolamine;DPPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamine imidazole (DSPEI), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphate (18:1) PA; DOPA), ammonium bis((S)-2-hydroxy-3-(oleoyloxy)propyl) phosphate; 18:1 DMP; LBPA), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol); DOPI; 18:1 PI), 1,2-distearoyl-sn-glycero-3-phospho-L-serine; 18:0 PS), 1,2-dilinoleoyl-sn-glycero-3-phospho-L-serine (18:2 PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (16:0-18:1 PS; POPS), 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine;18:0-18:1 PS), 1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (18:0-18:2 PS), 1-oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:1 Lyso PS), 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:0 Lyso PS), and sphingomyelin.

24. A lipid nanoparticle (LNP) comprising: (A) an ionizable lipid, wherein the ionizable lipid is a compound of any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof; and (B) an RNA encoding a compound.

25. The LNP of claim 24 further comprises: (a) PEG-lipids; (b) structural lipids; and (c) non-ionizable lipids and / or zwitterionic lipids.

26. The LNP of claim 25, wherein the PEG-lipid is selected from the group consisting of: PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC and PEG-DSPE.

27. The LNP of claim 25, wherein the structural lipid is selected from the group consisting of: cholesterol, coprosterol, phytosterol, ergosterol, campesterol, stigmasterol, phytosterol, ursolic acid, and α-tocopherol.

28. The LNP of claim 25, wherein the non-ionizable lipid is a phospholipid selected from the group consisting of: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE ... 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DMPC) hocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (1-palmitoyl-2-oleyl-2 ... yl-2-oleoyl-sn-glycero-3-phosphocholine; POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine; 18:0 diether PC), 1-oleoyl-2-cholesterylhemisuc cinoyl-sn-glycero-3-phosphocholine; OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine;C16 Lyso PC), 1,2-dilinalinyl-sn-glycero-3-phosphocholine, 1,2-diarachidonicyl-sn-glycero-3-phosphocholine, 1,2-bis(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-diphytanoylsn-glycero-3-phosphoethanolamine (ME 16.0) PE), 1,2-distearyl-sn-glycero-3-phosphate ethanolamine, 1,2-dilinoleyl-sn-glycero-3-phosphate ethanolamine, 1,2-dilinoleyl-sn-glycero-3-phosphate ethanolamine, 1,2-diarachidonicyl-sn-glycero-3-phosphate ethanolamine, 1,2-bis(docosahexaenoyl)-sn-glycero-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphate sodium salt (1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt) Sodium (S)-2-ammonium-3-((((R)-2-(oleyloxy)-3-(stearyloxy)propoxy)oxophosphatidyl)oxy)propionate (L-α-phosphatidylinosine; brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylcholine (DMPC) Phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (1,2-dioleoyl-sn-glycero-3-(phospho-L-serine; DOPS), acell-fusogenic phospholipid (DPhPE), dipalmitoylphosphatidylethanolamine;DPPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamine imidazole (DSPEI), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphate (18:1) PA; DOPA), ammonium bis((S)-2-hydroxy-3-(oleoyloxy)propyl) phosphate; 18:1 DMP; LBPA), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol); DOPI; 18:1 PI), 1,2-distearoyl-sn-glycero-3-phospho-L-serine; 18:0 PS), 1,2-dilinoleoyl-sn-glycero-3-phospho-L-serine (18:2 PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (16:0-18:1 PS; POPS), 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine;18:0-18:1 PS), 1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (18:0-18:2 PS), 1-oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:1 Lyso PS), 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:0 Lyso PS), and sphingomyelin.

29. The LNP of request item 25, wherein the encoding RNA is mRNA.

30. The LNP of request item 25, wherein the coding RNA is a circular RNA (circRNA).

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