Alexandra L. Howell

MicroRNAs (miRNAs) are a class of small non-coding RNAs, 19 to 24 nucleotides in length, that regulate a range of developmental and physiological processes by modulating gene expression through pos...

Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. CasX2 (PlmCas12e) belongs to the class II CRISPR system derived from Planctomycetes, a non-pathogenic bacterium present in aquatic and terrestrial soils and offers several advantages as a potential therapeutic CRISPR system over Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9). These advantages include its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and the absence of pre-existing immunity in humans. We compared the cleavage efficiency and double-stranded break repair characteristics between CasX2 and CasX2^Max, a recently generated CasX2 variant with three amino acid substitutions, for targeting CCR5, a gene that encodes the CCR5 receptor important for HIV-1 infection. Two single guide RNAs (sgRNAs) were designed that flank the 32 bases deleted in the natural CCR5 ∆32 mutation. Nanopore sequencing demonstrated that CasX2 using sgRNAs with spacers of 17 nucleotides (nt), 20 nt or 23 nt in length were ineffective at cleaving genomic CCR5. In contrast, CasX2^Max using sgRNAs with 20 nt and 23 nt spacer lengths, enabled cleavage of genomic CCR5. Structural modelling indicated that two of the CasX2^Max amino acid substitutions enhanced sgRNA-DNA duplex stability, while the third improved DNA strand alignment within the catalytic site. These structural changes likely underlie the increased activity of CasX2^Max in cellular gene excision. In sum, CasX2^Max consistently outperformed native CasX2 across all assays and represents a superior gene-editing platform for therapeutic applications.

Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. We evaluated CasX2 ( Plm Cas12e), a class II CRISPR system derived from Planctomycetes , a non-pathogenic bacterium present in aquatic and terrestrial soils. CasX2 offers several advantages over Streptococcus pyogenes Cas9 ( Sp Cas9) and Staphylococcus aureus Cas9 ( Sa Cas9), including its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and no known pre-existing immunity in humans. A recent study reported that a three amino acid substitution in CasX2 significantly enhanced cleavage activity (1). Therefore, we compared cleavage efficiency and double-stranded break repair characteristics between the native CasX2 and the variant, CasX2 ^Max , for cleavage of CCR5 , a gene that encodes the CCR5 receptor important for HIV-1 infection. Two CasX2 single guide RNAs (sgRNAs) were designed that flanked the 32 bases deleted in the natural CCR5 Δ32 mutation. Nanopore sequencing demonstrated that CasX2 using sgRNAs with spacers of 17 nucleotides (nt), 20 nt or 23 nt in length were ineffective at cleaving genomic CCR5. In contrast, CasX2 ^Max using sgRNAs with 20 nt and 23 nt spacer lengths, enabled robust genomic cleavage of CCR5 . Structural modeling indicated that two of the CasX2 ^Max substitutions enhanced sgRNA-DNA duplex stability, while the third improved DNA strand alignment within the catalytic site. These structural changes likely underlie the increased activity of CasX2 ^Max in cellular gene excision. In sum, CasX2 ^Max consistently outperformed native CasX2 across all assays and represents a superior gene-editing platform for therapeutic applications.

Background: MicroRNAs (miRNAs) are increasingly being investigated as potential biomarkers for child development and disease. Although a growing number of studies are utilizing infant and toddler stool for transcriptomic analyses, no studies have compared protocols for preserving and extracting miRNAs from this specimen type, despite unique challenges, including abundant levels of RNAses and microbial RNA. Methods: To address this, we first compared three commercially available kits and four preservation methods for their ability to yield high quality RNA from infant and toddler stool (Phase 1). RNA quality was determined by fragment analyzer. Results: Of the three RNA extraction kits compared, Zymo BIOMICs yielded the highest overall RNA Quality Number (RQN) (median (range) RQN 9.4 (5.7-10.0)). Of the four preservation methods tested, stool collected in RNAlater and Zymo DNA/RNA Shield Fecal Collection Tubes yielded the highest two RQNs (median (range) RQN 9.8 (5.7-10.0) and 9.4 (5.4-10.0), respectively), which did not differ significantly from each other ( p = 0.47). Second, using miRNA-seq we directly compared miRNA profiles for RNA extracted using the Zymo BIOMICs kit from paired aliquots of the same stool sample from four infants collected into RNAlater and Zymo DNA/RNA Shield Fecal Collection Tubes (Phase 2). Given that microbial sequences greatly outnumber human miRNAs in stool, reads were first classified as human versus microbial prior to aligning human-classified reads to miRBase v22.1. The percentage of reads classified as human and the percentage of human reads aligning to miRBase did not differ for samples collected in RNAlater versus Zymo Shield ( p = 0.12 and p = 0.86, respectively). Furthermore, after multiple testing correction, normalized miRNA counts did not differ significantly between the two preservatives for any of the 42 human miRNAs detected across the eight samples. Conclusions: Collecting infant and toddler stool in either RNAlater or Zymo DNA/RNA Shield Fecal Collection Tubes, when paired with RNA extraction using the Zymo BIOMICs extraction kit, yielded high-quality RNA with similar human miRNA profiles. Moreover, of the 42 miRNAs that were detected, several (i.e., miR-194a-3p, miR-200c-3p, miR-26a-5p) are thought to contribute to overall gut homeostasis. These findings may inform protocols for future studies that aim to profile miRNAs in infant and toddler stool to evaluate their potential utility as biomarkers for children’s health.

CRISPR/Cas is under development as a therapeutic tool for the cleavage, excision, and/or modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9), alternative CRISPR systems have been identified using metagenomic datasets from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of gene editors. The Cas12e (CasX1, CasX2) endonucleases from non-pathogenic Deltaproteobacteria (DpeCas12e) and Planctomycetes (PlmCas12e) are more compact than SpCas9, have a more selective protospacer adjacent motif (PAM) requirement, and deliver a staggered cleavage cut with 5-7 base overhangs. We investigated varying guide RNA (spacer) lengths and alternative PAM sequences to determine optimal conditions for PlmCas12e cleavage of the cellular gene CCR5 (CC-Chemokine receptor-5). CCR5 encodes one of two chemokine coreceptors required by HIV-1 to infect target cells, and a mutation of CCR5 (delta-32) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently, CCR5 has been an important target for gene editing utilizing CRISPR, TALENs, and ZFNs. We determined that CCR5 cleavage activity varied with the target site, guide RNA length, and the terminal nucleotide in the PAM sequence. Our analyses demonstrated a PlmCas12e PAM preference for purines (A, G) over pyrimidines (T, C) in the fourth position of the CasX2 PAM (TTCN). These analyses have contributed to a better understanding of CasX2 cleavage requirements and will position us more favorably to develop a therapeutic that creates the delta-32 mutation in the CCR5 gene in hematopoietic stem cells.

Gene editing approaches using CRISPR/Cas9 are being developed as a means for targeting the integrated HIV-1 provirus. Enthusiasm for the use of gene editing as an anti-HIV-1 therapeutic has been tempered by concerns about the specificity and efficacy of this approach. Guide RNAs (gRNAs) that target conserved sequences across a wide range of genetically diverse HIV-1 isolates will have greater clinical utility. However, on-target efficacy should be considered in the context of off-target cleavage events as these may comprise an essential safety parameter for CRISPR-based therapeutics. We analyzed a panel of Streptococcus pyogenes Cas9 ( Sp Cas9) gRNAs directed to the 5′ and 3′ long terminal repeat (LTR) regions of HIV-1. We used in vitro cleavage assays with genetically diverse HIV-1 LTR sequences to determine gRNA activity across HIV-1 clades. Lipid-based transfection of gRNA/Cas9 ribonucleoproteins was used to assess targeting of the integrated HIV-1 proviral sequence in cells ( in vivo ). For both the in vitro and in vivo experiments, we observed increased efficiency of sequence disruption through the simultaneous use of two distinct gRNAs. Next, CIRCLE-Seq was utilized to identify off-target cleavage events using genomic DNA from cells with integrated HIV-1 proviral DNA. We identified a gRNA targeting the U3 region of the LTR (termed Sp Cas9-127 _HBX2 ) with broad cleavage efficiency against sequences from genetically diverse HIV-1 strains. Based on these results, we propose a workflow for identification and development of anti-HIV CRISPR therapeutics.

Immune response modifiers are being studied as therapeutic agents for viral infections and cancer. These molecules include agonists for the Toll-like receptors (TLR), a family of innate immune receptors. TLR7 and 8, located in cellular endosomes, bind single-stranded RNA characteristic of viral genomes, and trigger intracellular signaling pathways that induce inflammatory cytokines and antiviral innate immune factors. We studied the anti-HIV-1 effects of gardiquimod, a specific TLR7 agonist when used at concentrations below 10 μM, in macrophages and activated peripheral blood mononuclear cells (PBMCs). Gardiquimod, added prior to or within 2 days after infection with X4, R5, or dual-tropic (R5/X4) strains of HIV-1, significantly reduced infection in these cells. Cocultures of activated PBMCs added to gardiquimod-treated and HIV-1-exposed macrophages demonstrated minimal HIV-1 replication for up to 10 days, suggesting that gardiquimod inhibited activated PBMCs viral amplification from HIV-1-exposed macrophages. Gardiquimod treatment of both activated PBMCs and macrophages induced interferon-alpha (IFN-α) transcription within hours of addition, and sustained IFN-α protein secretion for several days. Treatment of cells with a peptide inhibitor to the MyD88 adaptor protein blocked the induction of IFN-α by gardiquimod, and partially reversed the anti-HIV effects in activated PBMCs. Blocking the IFN-α receptor with a neutralizing antibody also reduced the anti-HIV effect of gardiquimod. Gardiquimod inhibited HIV-1 reverse transcriptase, an early step in the life cycle of HIV-1. These findings suggest that gardiquimod, functioning as both an immune system modifier and a reverse transcriptase inhibitor, could be developed as a novel therapeutic agent to block systemic and mucosal transmission of HIV-1.

Human Immunodeficiency Virus-type 1 (HIV- 1) binds to CD4 and CCR5 receptors on target cells in the human female reproductive tract. We sought to determine whether reducing levels of messenger RNA (mRNA) transcripts that encode these receptors in female reproductive tract cells could protect mucosal tissue explants from HIV- 1 infection. Explants prepared from the endometrium, endocervix, and ectocervix of hysterectomy tissues from HIV-1 sero-negative women were exposed to nanoparticles containing CD4- and CCR5-specific short-interfering RNA (siRNA) sequences. Explants were then exposed two days later to HIV-1, and HIV-1 reverse transcripts were measured five days post-infection. Explants treated with nanoparticles containing CD4- and CCR5-specific siRNA showed reduced levels of CD4 and CCR5 transcripts, and significantly lower levels of HIV-1 reverse transcripts compared to those treated with an irrelevant siRNA. In female reproductive tract explants and in peripheral blood cell cultures, siRNA transfection induced the secretion of IFN-alpha (IFN-α), a potent antiviral cytokine. In female mice, murine-specific Cd4-siRNA nanoparticles instilled within the uterus significantly reduced murine Cd4 transcripts by day 3. Our findings demonstrate that siRNA nanoparticles reduce expression of HIV-1 infectivity receptors in human female reproductive tract tissues and also inhibit HIV-1 infection. Murine studies demonstrate that nanoparticles can penetrate the reproductive tract tissues in vivo and silence gene expression. The induction of IFN-α after siRNA transfection can potentially contribute to the antiviral effect. These findings support the therapeutic development of nanoparticles to deliver siRNA molecules to silence host cell receptors in the female reproductive tract as a novel microbicide to inhibit mucosal HIV-1 transmission.

The human immunodeficiency virus type 1 (HIV-1) infects cells within mucosal tissues, including those of the female reproductive tract (FRT). The mechanism of viral transmission within the FRT and the mode of viral spread to the periphery are not completely understood. We performed phenotypical analyses and infectivity studies of primary FRT cells to identify potential targets of infection within the FRT. Our findings indicate that expression of HIV-1 receptors and coreceptors in the FRT varies as a function of menstrual-cycle stage, suggesting that sex hormone levels may influence a woman’s susceptibility to infection by HIV-1. Moreover, HIV-1 strains that utilize the CXCR4 chemokine receptor for infectivity are able to undergo reverse transcription, integration, viral DNA transcription, and viral release, whereas viral strains that utilize CCR5 do not undergo these early replicative events, and are only released unmodified from these cells. This indicates that several mechanisms for viral infection and transmission are present throughout the FRT.