Patent Publication Number: US-2021171952-A1

Title: Treating herpesvirus-mediated intestinal dysfunction for prevention of age-related neurodegeneration

Description:
BACKGROUND 
     Infection with one or more human herpesviruses (HHVs) is ubiquitous in most populations by the eighth decade of life. HHVs primarily enter the body through trigeminal ganglia or olfactory neurons. Virions shed from these neurons can enter circulating monocytes and dendritic precursors. Circulating monocytes and dendritic precursors differentiate when they encounter inflammatory cytokines. Phagocytes and dendritic cell differentiation reactivate HHV from latency. HHVs mostly remain latent through the first seven decades, apart from replicating reservoirs. The frequency of HHV reactivation depends on the HHV strain and host variables, such as stress, age, and genetics. For instance, cytomegalovirus (HCMV) is an HHV that remains latent but reactivates in individuals experiencing stress, depressed immunity, or in healthy individuals over the age of 70. Genetic variants such as polymorphisms in type 3 interferon (IFNL3/4) affect susceptibility to HCMV activation. 
     There are eight HHVs and each can infect cells residing in the intestinal wall or tract. HCMV/HHV5 replicates in epithelial, fibroblast, endothelial, and dendritic cells. The Epstein-Barr virus (EBV/HHV4) infects B lymphocytes. The roseolovirus (HHV 6/7 ) causes roseola infantum, and replicates in peripheral blood mononuclear cells (PBMC), NK cells, and T lymphocytes. Herpes simplex (Hsv1/2-HHV1/2) and varicella zoster virus (VZV-HHV3) replicate in fibroblast, epithelial cells, and sensory neurons. 
     HHV infections could contribute to developing Alzheimer&#39;s disease (AD), Parkinson&#39;s disease, Lewy body dementia, vascular dementia, frontotemporal dementia, and amyotrophic lateral sclerosis. Any causal relationship between HHV infection and neurodegeneration is still unsettled because some studies have reported no association, while those that have cannot prove causation. However, studies measuring viral DNA from post-mortem brain tissue may have failed to detect the virus. For instance, congenital HCMV infections with severe brain degeneration show a conspicuous absence of viral DNA, which suggests that a non-cell autonomous bystander effect mediates significant pathology. This scenario is reminiscent of the HIV-associated neurocognitive disorder mediated by the HIV protein TAT. Association by seropositivity is also wrought with error. Seronegative subjects can test PCR positive for the HCMV DNA in blood and seropositive subjects can consistently test PCR negative for HCMV DNA. HCMV can also reactivate intermittently in short intervals, so blood HCMV DNA might be negative one week and positive the next. Thus, researchers may have erroneously concluded no association exists between some HHVs and neurodegeneration. Further, like HCMV, other HHVs infect immune cells, and their infection status is associated with oxidative stress, shortened telomeres of lymphocytes, reduction of B cell lineages, and T-cell senescence. The subsequent effects of HHV infection on immunity may increase host susceptibility to other opportunistic infections, which are increasingly being associated with neurodegeneration. 
    
    
     DETAILED DESCRIPTION 
     Neurodegeneration is also associated with gut microbiota dysbiosis. HHV infection of B lymphocytes, T-cells, dendritic cells, and PBMCs can compromise intestinal immunity and increase pathogen-mediated inflammation. Increased inflammation is associated with gut microbiota dysbiosis, whereby the prevalence of healthy symbiotic bacteria is reduced, and pathogenic bacteria increased. Thus, it is not surprising that HHV infection is associated with colitis in healthy immunocompetent patients. HHV infection of intestinal epithelial cells, vascular endothelial cells, and fibroblasts may compromise the intestinal barrier and allow microbial translocation. 
     Oxidative stress related to HHV infection may accelerate stein cell senescence over time. Additionally, HHV is associated with impaired the Wnt/β-catenin signaling and loss of β-catenin-mediated gene transcription, which is required for epithelial cell specification, maintenance, and replication. Replicative senescence of transiently replicating epithelium and stein cells could result in decreased barrier function. Decreased barrier function, called “leaky gut”, could permit microbial translocation. Elderly with reduced immunity and blood-brain-barrier are at increased risk for microbes entering the brain. Microbial infection in the brain can initiate an inflammatory response that causes neurodegeneration. Further, with advanced age, the inflammatory response is more sustained. Inflammatory bowel diseases, such as Crohn&#39;s disease and ulcerative colitis, are associated with a leaky gut, and accordingly, are associated with an increased rate of Parkinson&#39;s disease. Genetic variants that increase the risk of Crohn&#39;s disease are also associated with Parkinson&#39;s disease. A microbial translocation etiology of Parkinson&#39;s disease is supported by the finding that mice injected with gut bacteria from Parkinson&#39;s disease patients develop neuronal α-synuclein clumping and behavioral symptoms. Further support of a gut-related etiology for Parkinson&#39;s disease comes from the finding that α-synuclein aggregates are found within the enteric nerve terminals of every Parkinson&#39;s disease patient examined. Parkinson&#39;s disease patients also have reduced occludin protein, a component of tight junctions, compared to healthy age-matched controls, which could disrupt the intestinal epithelial barrier. 
     A variety of translocated microbes could explain the heterogenous nature of neurodegeneration. Microbe phagocytosis by antigen-presenting cells selects an immunogen to initiate an immune response. If a host protein has a similar sequence to a microbial epitope it will cross-react and cause autoimmune disease by mimicry. For instance, a Uniprot blast of tau protein against bacterial proteasomes identified bacteria with high sequence similarity to proteins from bacterial strains of Actinomyces, Streptococcus, and Streptomyces. The α-synuclein protein, which forms aggregates in Parkinson&#39;s disease patients, has sequence-similarity to proteins found in fecal bacteria. A Uniprot blast of α-synuclein against microbial proteomes identified sequence similarity with the uncharacterized protein from the Verrucomicrobia bacterium, with 43.7% positives (2.3e-04 E-value), and with similarity to the chromosome segregation ATPase from Bifidobacterium, with 47% positives (1.4e-03 E-value). 
     Anti-a-synuclein IgG2 are significantly increased in the plasma from Parkinson&#39;s disease patients. IgG2 increases in response to bacterial infections and reacts to bacterial polysaccharides. Intracellular antibodies can cause target aggregation, which are both degraded by the proteasome. Proteasome degradation slows with aging, which could explain age-related onset of neurodegeneration. It is not known whether IgG physically associates with neurodegeneration related-protein aggregates. Accordingly, levels of antinuclear antibodies increase with age and are associated with signs of senescence, e.g. telomere shortening. Antinuclear antibodies, indicative for autoimmune disease, are also found with infectious disease and frequently with herpes simplex encephalitis. 
     A chronic subclinical HCMV infection in the colon could impair barrier function and allow microbial translocation. Interestingly, Parkinson&#39;s disease is associated with significantly reduced CD8+T cell senescence compared to age-matched control, which could result if CD8+T cells do not engage in restricting viral pathogens. Thus, while it appears that Parkinson&#39;s disease patients can mount an effective cytotoxic CD8+T immune response against invading microbes, it still requires professional antigen presenting cells, such as dendritic cells, which may be functionally compromised by Hcmv infection. 
     HHV infection has the potential to be a major cause of intestinal dysfunction. Hcmv-infected cultured human fibroblasts have markedly altered miRNA expression. Thus, in situ Hcmv-infected intestinal niche fibroblasts are likely to have altered exosome content, which may be deleterious to epithelial maintenance. Preventing intestinal HHV activity in intestinal cells can prevent inflammation, microbiota dysbiosis, barrier dysfunction, autoimmune disease, and subsequent neurodegeneration. HSV and VZV infection can be treated with nucleoside analogs acyclovir and penciclovir. Although, the nucleoside analogs are known treatments for HSV and VZV infection, HSV/VZV-mediated intestinal dysfunction is not recognized, and thus nucleoside treatment is not prescribed. It is highly likely that enteric sensory neurons harbor HSV and VZV, like the olfactory and trigeminal neurons, and that their reactivation contributes to intestinal inflammation and microbiota dysbiosis. The ability to treat intestinal dysfunction resulting from HSV/VZV-infected enteric neurons is not known. It is true that patients receiving nucleoside analogs for resolving herpetic lesions are inadvertently treated for intestinal dysfunction. However, unexpected success and long-sought need are secondary considerations supporting the non-obviousness of treating HSV/VZV-mediated intestinal dysfunction with nucleoside analogs. 
     Unfortunately, long term use of nucleoside analogs can result in HSV and VZV thymidine kinase mutations rendering the virus nucleoside resistant. Except for human cytomegalovirus (Hcmv), there is no treatment for other HHVs. Hcmv antivirals have serious side-effects and result in resistance, so they are not viable long-term options. 
     A novel method for preventing HHV-activity is to deliver exosomes containing stock and customized antiviral, antioxidant, replication-inducing, and anti-inflammatory factors that will be administered by oral dosing for intestinal delivery. Exosomes are isolated by ultracentrifugation from modified human colonic fibroblast (ATCC-49). The content of colonic fibroblast exosomes contains therapeutic factors for intestinal epithelial cells. For instance, exosomes from a colon fibroblast cell line carry amphiregulin, which binds epidermal growth factor receptor, and rescues intestinal epithelial cells from cell death in organoid culture. Exosome content from cultured fibroblasts will be manipulated by inserting donor DNA that encode pre-miRNAs, mRNA, or long non-coding RNA (lncRNA) into the fibroblast genome using the Sleeping Beauty (SB) transposon system. Construct expression can be driven by fibroblast housekeeping promoters. 
     Combining intestinal exosome delivery with CRISPR gene therapy provides the ability to alter host and viral DNA and potentially eliminate viral infection. For instance, CRISPR has the potential to elicit site-specific cleavage at viral genomic sequences. Delivery of the Cas9 protein or novel iterations, along with a guide strand and an expression cassette, can safely target the insertion of antiviral transgenes. 
     Exogenous exosomes need to penetrate through the mucosal bacteria to reach epithelial cells and vascular endothelial cells. A new method to prevent and treat HHV-mediated inflammation, microbiota dysbiosis, barrier dysfunction, autoimmune disease, and subsequent neurodegeneration is to ingest recombinant bacteria that will survive among the gut flora and release exosomes containing antiviral factors. The type of bacteria selected is critical to therapeutic efficacy. 
     The bacteria  Clostridium, Lactobaccillus, Enterococcus,  and  Akkermansia  are associated with the mucosal surface, and therefore have the proximity to deliver high concentrations of exosomes with antiviral factors to intestinal cells. Bacteria can be engineered to express antiviral factors for exosome packaging and secretion. A DNA construct encoding miRNA, mRNA, and oligonucleotides can be integrated stably into the bacterial genomes by site-specific recombination. 
     Expression constructs are designed to express select miRNA/shRNA, mRNA, lncRNA, and protein as exosome cargo that reduce HHV activity, oxidative stress, and inflammation as well as increase replicative capacity of intestinal epithelial and vascular endothelial cells. The following is an example of the antiviral factors to be delivered in exosomes. Human antiviral miRNAs proven to reduce HHV replication include SEQ ID NO:1 hsa-miR-324-5p, SEQ ID NO:2 hsa-miR-185, SEQ ID NO:3 hsa-miR-29b, SEQ ID NO:4 hsa-miR-1287, SEQ ID NO:5 hsa-miR-199a-3p, SEQ ID NO:6 hsa-miR-214, SEQ ID NO:7 hsa-miR-21, SEQ ID NO:8 hsa-miR-200b-3p, SEQ ID NO:9 hsa-miR-200c-3p, SEQ ID NO:10 hsa-miR221, and SEQ ID NO:11 hsa-miR-24-1. Proteins involved in antiviral defense, such as SEQ ID NO:12 Sp100, SEQ ID NO:13 Daxx, SEQ ID NO:14 PML, SEQ ID NO:15 BclAF1, SEQ ID NO:16 Tetherin/Bts-2, SEQ ID NO: 17 Trim5-alpha, and SEQ ID NO:18 Apobec-3G could be delivered as mRNA or protein. For instance, Daxx directs HDAC to the major immediate early gene promoter to silence viral transcription, and BclAF1 is critical for interferon 1 response; both proteins are degraded by HHV. 
     Aside from the autosomal dominant mutations causing early-onset AD, apolipoprotein E (ApoE) allelic variants explain the highest variance in AD risk. The ApoE4 allele confers the greatest AD risk, with ApoE3 neutral, and ApoE2, a protective variant. The explanation for higher AD risk with ApoE4 could be related to higher intracellular cholesterol content, especially in lipid rafts, which would increase virion binding affinity. However, another explanation is that ApoE2/E3 has less affinity for LDL or LDLR than ApoE4 and may be more available to bind viral glycoproteins both inside and outside the cell. Accordingly, only the LDLR and LDL binding domains of ApoE3 can restrict HIV infectivity. The LDLR domain can even protect against early-onset AD from the autosomal dominant mutation, PSEN1, in patients homozygous for the ApoE3-Christchurch mutation (R136S). Moreover, ApoE2/3 is more concentrated in serum than ApoE4. Therefore, one embodiment of the invention includes delivering SEQ ID NO:19 ApoE2 or SEQ ID NO: 20 ApoE3-Christchurch protein or LDLR domain to intestinal cells via exosomes to protect against HHV infectivity. 
     Another embodiment of the invention includes short hairpin RNAs (shRNA) designed to silence HHV gene expression. For instance, a shRNA targeting the SEQ ID NO:21 HCMV UL83 gene (pp65) at the sequence GCAAGATCTCGCACATCATGC will reduce pp65 protein expression and release its inhibition on innate and adaptive immunity. Targeting the SEQ ID NO:22 smallest capsid protein at the sequence GCGCATGTCCAGTCTGTTTAA can reduce HCMV yield by 10,000-fold. Additional shRNAs designed to restrict HHV gene expression will be included in the fibroblast and bacteria expression constructs. 
     HHV infection induces oxidative stress. For instance, HCMV increases NAD(P)H oxidase (NOX) activity in endothelial cells and NOX4 expression is increased in vascular endothelial cells from Alzheimer&#39;s patients. NOX4 expression can be inhibited by SEQ ID NO:23 hsa-miR-137 and SEQ ID NO:24 hsa-miR99a. Messenger RNAs encoding proteins that reduce oxidative stress include SEQ ID NO:25 superoxide dismutase, SEQ ID NO:26 glutathione reductase, and the protein SEQ ID NO:27 Hic-5, which inhibits NOX4 activity by promoting the ubiquitin-proteasome degradation of NOX4. 
     Intestinal inflammation can be reduced by delivering exosome factors to immune cells. For instance, SEQ ID NO:28 hsa-miR-219a-5p delivered to T cells downregulates Th1/Th17 cell differentiation. Delivery of SEQ ID NO:29 hsa-miR-146a, SEQ ID NO:30 hsa-miR-19b, SEQ ID NO:31 hsa-miR-590-5p, and SEQ ID NO:32 miR-495 reduces inflammatory cell injury. Delivery of the lncRNA SEQ ID NO: 33 Mirt2 to dendritic cells upregulates IL-22 expression and helps resolve chronic inflammation. 
     HCMV may control replicative senescence of epithelial and endothelial cells. For instance, HCMV infection increases DKK1 expression. DKK1 interacts with Wnt co-receptors LRP5/6, leading to the degradation of β-catenin. Degradation of β-catenin prevents the Wnt pathway signaling required for epithelial cell homeostasis and renewal. The EBV miRNA, -SEQ ID NO: 34 EBV-miR-BART10-3p, reduces DKK1 protein expression by 60%. To prevent oncogenic potential, an inducible promoter can drive miR-BART10-3p expression in recombinant bacteria. 
     To ensure that miRNAs, mRNA, lncRNA, and protein are sorted to the exosome, the donor DNA will include a 3′ export motif, GGAG or GGCU, for hnRNPA2B1 and SYNCRIP processing, respectively. Messenger RNA encoding protein will be directed to the exosome by including a fused ubiquitylated-coding sequence (CTGCC) for ECSRT-dependent processing or a CD63 association sequence for ECSRT-independent processing. 
     Exosomes will be isolated by ultracentrifugation or serial filtration to characterize content. Select combinations of exosome cargo will be tested for their ability to reduce HHV-activity, oxidative stress, inflammation, and induce replication in columnar intestinal epithelial cell cultures. With optimal exosome cargo defined, recombinant fibroblast cultures will be scaled for mass production of therapeutic exosomes. 
     HHV strains can have different cell tropism, so activity in one replicating reservoir may not accurately reflect activity in another reservoir. Currently, intestinal HHV activity is measured by immunolabeling biopsy specimens or detecting HHV nucleic acid by PCR. However, a biopsy procedure is costly and HHVs can produce significant amounts of capsids or dense bodies without infectious DNA. Thus, to quickly and economically detect intestinal HHV activity, HHV proteins will be measured in stool by ELISA. High copy tegument proteins are particularly amenable to detection by immunoassay, but signal amplification may be required. Examples of tegument proteins to be measured are SEQ ID NO:35 VP22-HHV1/2/3/4, SEQ ID NO: 36 pp65-HHV4/5/6, and SEQ ID NO: 37 pp150-HHV4/5/6. Epitopes that are shared among HHV types are selected to estimate total intestinal HHV load. To determine if exosome therapy is required, stool HHV protein levels can be measured during immunosuppression, stress, and monitored periodically after age 60. 
     HHVs reactivate during immunosuppression, such as with transplant or chemotherapy, and cause life-threatening disease. The described novel treatments are useful for restricting HHV-mediated intestinal disease and could be administered prophylactically in immunosuppressed patients. 
     SEQUENCE DISCLOSURE 
     The sequences are disclosed in a 79KB text file named, “10001B-US-NP_Sequences-as-filed”, created on 2020-03-25.