Delivering crispr therapeutics with lipid nanoparticles

Abstract

The present invention provides lipid particles comprising therapeutic nucleic acids such as gRNA that target gene expression.

1 . A nucleic acid-lipid particle comprising:
 (a) one or more guide RNA (gRNA) sequences that comprise a first sequence that corresponds to a target sequence and a second sequence that is a tracer RNA sequence located 3′ of the first sequence;   (b) a cationic lipid; and   (c) a non-cationic lipid.   
     
     
         2 . The nucleic acid-lipid particle of  claim 1 , which further comprises a mRNA sequence encoding a CRISPR associated protein 9 (Cas9). 
     
     
         3 . The nucleic acid-lipid particle of  claim 2 , wherein the mRNA sequence encoding the Cas9 further comprises a sequence encoding a nuclear localization signal (NLS). 
     
     
         4 . (canceled) 
     
     
         5 . The nucleic acid-lipid particle of  claim 2 , wherein the mRNA sequence encoding the Cas9 further comprises a polyA tail, a 5′ untranslated region (UTR) and/or a 3′ UTR. 
     
     
         6 . The nucleic acid-lipid particle of  claim 1 , wherein the cationic lipid is selected from the group consisting of 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA; Compound (15)), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine (DLin-MP-DMA; Compound (8)), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethyl amino)butanoate) (Compound (7)), (6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl 5-(dimethylamino)pentanoate (Compound (13)), a salt thereof, and a mixture thereof. 
     
     
         7 . The nucleic acid-lipid particle of  claim 1 , wherein the non-cationic lipid is cholesterol or a derivative thereof. 
     
     
         8 . The nucleic acid-lipid particle of  claim 1 , wherein the non-cationic lipid is a phospholipid. 
     
     
         9 . The nucleic acid-lipid particle of  claim 1 , further comprising a conjugated lipid that inhibits aggregation of particles. 
     
     
         10 . The nucleic acid-lipid particle of  claim 9 , wherein the conjugated lipid that inhibits aggregation of particles is a polyethyleneglycol (PEG)-lipid conjugate. 
     
     
         11 . The nucleic acid-lipid particle of  claim 10 , wherein the PEG-lipid conjugate is selected from the group consisting of a PEG-diacylglycerol (PEG-DAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, a PEG-phospholipid conjugate, a PEG-ceramide (PEG-Cer) conjugate, and a mixture thereof. 
     
     
         12 . The nucleic acid-lipid particle of  claim 10 , wherein the PEG-lipid conjugate is PEG2000-C-DMA. 
     
     
         13 . (canceled) 
     
     
         14 . The nucleic acid-lipid particle of  claim 1 , wherein the gRNA is fully encapsulated in the particle. 
     
     
         15 . The nucleic acid-lipid particle of  claim 1 , wherein the particle has a total lipid:gRNA mass ratio of from about 5:1 to about 15:1. 
     
     
         16 . (canceled) 
     
     
         17 . The nucleic acid-lipid particle of  claim 1 , wherein the particle has an electron dense core. 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . The nucleic acid-lipid particle of  claim 9 , wherein the conjugated lipid that inhibits aggregation of particles comprises from about 0.5 mol % to about 3 mol % of the total lipid present in the particle. 
     
     
         21 . (canceled) 
     
     
         22 . The nucleic acid-lipid particle of  claim 1  comprising two different gRNA sequences. 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . A pharmaceutical composition comprising a nucleic acid-lipid particle of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         28 . (canceled) 
     
     
         29 . A method for silencing expression of a target gene in a cell, the method comprising the step of contacting a cell comprising an expressed target gene with the nucleic acid-lipid particle of  claim 1  under conditions to silence the expression of the gene within the cell. 
     
     
         30 . (canceled) 
     
     
         31 . (canceled) 
     
     
         32 . (canceled) 
     
     
         33 . (canceled)