Gene editing-based method of attenuating the beta-amyloid pathway
Described herein is a method for treating Alzheimer's disease (AD) by selective silencing of the amyloid precursor protein (APP) using Cas9 nuclease gene editing. Methods of making and using genetic constructs comprising a Cas9 nuclease and a sequence encoding guide RNA (gRNA) specific to APP capable of truncating the C-terminus of APP, as well as compositions comprising these constructs, are provided.
1. A genetic construct encoding a Cas9 nuclease/gRNA ribonucleoprotein comprising, i. a sequence encoding for a Cas9 nuclease; and ii. a sequence encoding a gRNA specific to amyloid precursor protein (APP), wherein the encoded Cas9 nuclease/gRNA ribonucleoprotein directs cleavage of an APP gene to provide a C-terminal truncated APP having a length of 659, 670, 676, or 686 amino acids relative to a human APP amino acid sequence identified in SEQ ID NO: 12 or a mouse amino acid sequence identified in SEQ ID NO: 14.
2. The genetic construct of claim 1 , wherein the genetic construct is packaged in a viral vector selected from the group consisting of a lentiviral vector and an adeno-associated viral (AAV) vector.
3. The genetic construct of claim 1 , wherein the genetic construct further comprises at least one neuron specific promoter.
4. The genetic construct of claim 3 , wherein the neuron specific promoter is selected from the group consisting of human synapsin 1 (hSyn1) promoter and mouse Mecp2 promoter (pMecp2).
5. The genetic construct of claim 1 , wherein the genetic construct further comprises an RNA Pol III promoter.
6. The genetic construct of claim 5 , wherein the RNA Pol III promoter is a U6 promoter.
7. The genetic construct of claim 1 , wherein the sequence of the gRNA is selected from the group consisting of SEQ ID NOs:1-10.
8. The genetic construct of claim 1 , wherein the sequence of the Cas9 nuclease consists of SEQ ID NO:15.
9. The genetic construct of claim 1 , wherein the genetic construct comprises the sequence of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20.
10. A composition encoding a Cas9 nuclease/gRNA ribonucleoprotein comprising a first AAV vector comprising a sequence encoding for a Cas9 nuclease and a second AAV vector comprising a sequence encoding a gRNA specific to amyloid precursor protein (APP), wherein the encoded Cas9 nuclease/gRNA ribonucleoprotein directs cleavage of an APP gene to provide C-terminal truncated APP having a length of 659, 670, 676, or 686 amino acids relative to a human APP amino acid sequence identified in SEQ ID NO:12 or a mouse APP amino acid sequence identified in SEQ ID NO:14.
11. The composition of claim 10 , further comprising a nanocarrier delivery vehicle.
12. The composition of claim 11 , for delivery intravenously or intrathecally.