Compositions for the inactivation of virus replication and methods of making and using the same
Inventors
Cullen, Bryan R. • Kennedy, E. Matthew • Bogerd, Hal P. • Kornepati, Anand • Mefferd, Adam • Schinazi, Raymond F.
Assignees
Emory University • US Department of Veterans Affairs • Duke University
Publication Number
US-10286084-B2
Publication Date
2019-05-14
Expiration Date
2035-02-18
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Abstract
Provided herein are recombinant constructs, vectors and expression cassettes including a first promoter which is suitably a tRNA promoter operably connected to a first polynucleotide encoding a first single guide RNA and a second promoter operably connected to a second polynucleotide encoding a Cas9 polypeptide. The first single guide RNA includes a first portion complementary to a strand of a target sequence of a DNA virus and a second portion capable of interacting with the Cas9 polypeptide. Also provided are codon optimized Staphylococcus aureus derived Cas9 polynucleotides and polypeptides with nuclear localization signals and optionally an epitope tag. Also provided are constructs for production of sgRNAs including a tRNA. Methods of inhibiting viral replication, inhibiting expression of a target sequence from a virus or treating a viral infection or viral induced cancer using the compositions are also provided.
Core Innovation
The invention provides recombinant constructs, vectors, and expression cassettes comprising a first promoter connected to a polynucleotide encoding a first single guide RNA (sgRNA) and a second promoter connected to a polynucleotide encoding a Cas9 polypeptide, particularly derived from Staphylococcus aureus (Sau). The sgRNA contains a portion complementary to a DNA virus target sequence and a portion capable of interacting with the Cas9 polypeptide. Codon-optimized Sau Cas9 polynucleotides and polypeptides with nuclear localization signals and optional epitope tags are also provided. Further, constructs for sgRNA production including tRNA promoters are disclosed to facilitate efficient production using small promoter elements.
The method involves delivering these recombinant constructs or vectors into cells infected with DNA viruses, which include viruses classified in families such as hepadnaviridae, herpesviridae, papillomaviridae, and retroviridae. Upon expression, the sgRNA directs the Cas9 nuclease to cleave target viral DNA sequences, causing double-strand breaks that result in reduced gene expression, inhibition of viral replication, mutagenesis, or loss of the viral genome from cells.
The background problem addressed is that current gene therapy approaches using targeted DNA endonucleases like zinc finger nucleases (ZFNs) and TALENs face limitations including low specificity, difficulty in targeting multiple sites simultaneously, and large size of Cas9 proteins like Spy Cas9, which exceeds adeno-associated virus (AAV) packaging limits. Existing treatments for persistent DNA virus infections are often ineffective in fully clearing viral genomes such as covalently closed circular DNA (cccDNA) in hepatitis B virus (HBV) infection or latent herpes simplex virus (HSV). The compositions provided aim to enable multiplex targeting with improved specificity, facilitate delivery through smaller Cas9 proteins compatible with AAV vectors, and promote viral genome cleavage leading to viral inactivation or elimination.
Claims Coverage
The patent includes one independent claim directed to a recombinant Sau Cas9 construct and its use in viral targeting. The main inventive features cover the construct design, sgRNA arrangement, promoter selections, and viral vector embodiments.
Recombinant Sau Cas9 construct
A recombinant construct comprising a promoter operably connected to a polynucleotide encoding a recombinant Sau Cas9 polypeptide including SEQ ID NO: 55.
Inclusion of expression regulatory elements
The construct may include a poly(A) addition site, an intron to enhance expression, and a nuclear localization signal to direct Cas9 to the nucleus.
Promoter selection for Cas9 expression
Use of RNA Polymerase II dependent promoters/enhancers selected from EFS, hCMV, mCMV, CBA, hSynapsin, HSV TK, SV40 early, and LSP promoters.
Dual sgRNA expression with tRNA promoters
A second promoter operably linked to a second polynucleotide encoding a single guide RNA including a targeting portion complementary to a DNA viral sequence and a scaffold portion to interact with Cas9; the promoter can be U6 or smaller tRNA promoters such as Gln, Pro, Gly, Asn, Cys, Glu, or MHV68 tRNAs.
Targeting of DNA viruses with defined families
The sgRNA targets sequences in DNA viruses classified within hepadnaviridae, herpesviridae (including HSV-1, HSV-2, EBV, hCMV, VZV, KSHV), papillomaviridae, and retroviridae such as HIV-1.
Vector embodiments
Recombinant Sau Cas9 constructs can be incorporated into viral vectors selected from retrovirus, lentivirus, adenovirus, and adeno-associated virus (AAV) vectors, including single-AAV constructs harboring Cas9 and one or two sgRNAs.
The claims coverage consolidates the engineered recombinant Sau Cas9 constructs with expression elements and dual sgRNA cassettes under small promoters including tRNAs to enable packaging into AAV or other viral vectors for targeting viral DNA sequences from multiple virus families, thereby facilitating antiviral and anticancer gene therapy applications.
Stated Advantages
Multiplex targeting capability extends editing efficiency beyond single site cleavage, permitting gene disruption and full gene deletion.
Use of smaller Cas9 proteins such as Staphylococcus aureus-derived Cas9 allows packaging into preferred gene delivery vectors like AAV, overcoming delivery size limitations.
Efficient production of sgRNAs via tRNA promoters reduces vector size compared to conventional U6 promoters, facilitating inclusion of multiple sgRNAs in a single vector.
Targeted cleavage of viral DNA interferes with viral replication, reduces expression of viral genes, and can lead to elimination of viral episomal DNA including persistent viral genomes like HBV cccDNA and latent herpesvirus episomes.
The constructs enable specific treatment of viral infections and viral-induced cancers by inactivating essential viral genes and restoring tumor suppressor pathways in transformed cells.
Documented Applications
Treatment of viral infections caused by DNA viruses such as hepatitis B virus (HBV), herpes simplex virus types 1 and 2 (HSV-1, HSV-2), Epstein Barr Virus (EBV), human cytomegalovirus (hCMV), Varicella zoster virus (VZV), Kaposi's sarcoma associated herpesvirus (KSHV), human papillomavirus (HPV), and human immunodeficiency virus (HIV-1).
Elimination of viral episomal DNA genomes including HBV covalently closed circular DNA (cccDNA), latent herpesvirus episomes, and integrated or episomal HPV genomes in cancer cells.
Gene therapy applications for antiviral treatment using adeno-associated virus (AAV)-based vectors or other viral vectors delivering Cas9 and sgRNAs to infected tissues, including liver and neuronal tissue.
Treatment of viral induced cancers by targeting oncogenic viral genes such as HPV E6 and E7 to reactivate cellular tumor suppressors and induce arrest or apoptosis of transformed cells.
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