Tissue-specific biochemical differences between chronic wasting disease prions isolated from free-ranging white-tailed deer (Odocoileus virginianus).

Chronic wasting disease (CWD) is an invariably fatal prion disease affecting cervid species worldwide. Prions can manifest as distinct strains that can influence disease pathology and transmission. CWD is profoundly lymphotropic, and most infected cervids likely shed peripheral prions replicated in lymphoid organs. However, CWD is a neurodegenerative disease, and most research on prion strains has focused on neurogenic prions. Thus, a knowledge gap exists comparing neurogenic prions to lymphogenic prions. In this study, we compared prions from the obex and lymph nodes of naturally exposed white-tailed deer to identify potential biochemical strain differences. Here, we report biochemical evidence of strain differences between the brain and lymph node from these animals. Conformational stability assays, glycoform ratio analyses, and immunoreactivity scanning across the structured domain of the prion protein that refolds into the amyloid aggregate of the infectious prion reveal significantly more structural and glycoform variation in lymphogenic prions than neurogenic prions. Surprisingly, we observed greater biochemical differences among neurogenic prions than lymphogenic prions across individuals. We propose that the lymphoreticular system propagates a diverse array of prions from which the brain selects a more restricted pool of prions that may be quite different than those from another individual of the same species. Future work should examine the biological and zoonotic impact of these biochemical differences and examine more cervids from multiple locations to determine if these differences are conserved across species and locations.

Complement Regulatory Protein Factor H Is a Soluble Prion Receptor That Potentiates Peripheral Prion Pathogenesis.

Several complement proteins exacerbate prion disease, including C3, C1q, and CD21/35. These proteins of the complement cascade likely increase uptake, trafficking, and retention of prions in the lymphoreticular system, hallmark sites of early prion propagation. Complement regulatory protein factor H (fH) binds modified host proteins and lipids to prevent C3b deposition and, thus, autoimmune cell lysis. Previous reports show that fH binds various conformations of the cellular prion protein, leading us to question the role of fH in prion disease. In this article, we report that transgenic mice lacking Cfh alleles exhibit delayed peripheral prion accumulation, replication, and pathogenesis and onset of terminal disease in a gene-dose manner. We also report a biophysical interaction between purified fH and prion rods enriched from prion-diseased brain. fH also influences prion deposition in brains of infected mice. We conclude from these data and previous findings that the interplay between complement and prions likely involves a complex balance of prion sequestration and destruction via local tissue macrophages, prion trafficking by B and dendritic cells within the lymphoreticular system, intranodal prion replication by B and follicular dendritic cells, and potential prion strain selection by CD21/35 and fH. These findings reveal a novel role for complement-regulatory proteins in prion disease.

Endogenous Brain Lipids Inhibit Prion Amyloid Formation In Vitro.

The normal cellular prion protein (PrPC) resides in detergent-resistant outer membrane lipid rafts in which conversion to the pathogenic misfolded form is believed to occur. Once misfolding occurs, the pathogenic isoform polymerizes into highly stable amyloid fibrils. In vitro assays have demonstrated an intimate association between prion conversion and lipids, specifically phosphatidylethanolamine, which is a critical cofactor in the formation of synthetic infectious prions. In the current work, we demonstrate an alternative inhibitory function of lipids in the prion conversion process as assessed in vitro by real-time quaking-induced conversion (RT-QuIC). Using an alcohol-based extraction technique, we removed the lipid content from chronic wasting disease (CWD)-infected white-tailed deer brain homogenates and found that lipid extraction enabled RT-QuIC detection of CWD prions in a 2-log10-greater concentration of brain sample. Conversely, addition of brain-derived lipid extracts to CWD prion brain or lymph node samples inhibited amyloid formation in a dose-dependent manner. Subsequent lipid analysis demonstrated that this inhibitory function was restricted to the polar lipid fraction in brain. We further investigated three phospholipids commonly found in lipid membranes, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol, and found all three similarly inhibited RT-QuIC. These results demonstrating polar-lipid, and specifically phospholipid, inhibition of prion-seeded amyloid formation highlight the diverse roles lipid constituents may play in the prion conversion process.IMPORTANCE Prion conversion is likely influenced by lipid interactions, given the location of normal prion protein (PrPC) in lipid rafts and lipid cofactors generating infectious prions in in vitro models. Here, we use real-time quaking-induced conversion (RT-QuIC) to demonstrate that endogenous brain polar lipids can inhibit prion-seeded amyloid formation, suggesting that prion conversion is guided by an environment of proconversion and anticonversion lipids. These experiments also highlight the applicability of RT-QuIC to identify potential therapeutic inhibitors of prion conversion.

Pathways of Prion Spread during Early Chronic Wasting Disease in Deer.

Among prion infections, two scenarios of prion spread are generally observed: (i) early lymphoid tissue replication or (ii) direct neuroinvasion without substantial antecedent lymphoid amplification. In nature, cervids are infected with chronic wasting disease (CWD) prions by oral and nasal mucosal exposure, and studies of early CWD pathogenesis have implicated pharyngeal lymphoid tissue as the earliest sites of prion accumulation. However, knowledge of chronological events in prion spread during early infection remains incomplete. To investigate this knowledge gap in early CWD pathogenesis, we exposed white-tailed deer to CWD prions by mucosal routes and performed serial necropsies to assess PrPCWD tissue distribution by real-time quaking-induced conversion (RT-QuIC) and tyramide signal amplification immunohistochemistry (TSA-IHC). Although PrPCWD was not detected by either method in the initial days (1 and 3) postexposure, we observed PrPCWD seeding activity and follicular immunoreactivity in oropharyngeal lymphoid tissues at 1 and 2 months postexposure (MPE). At 3 MPE, PrPCWD replication had expanded to all systemic lymphoid tissues. By 4 MPE, the PrPCWD burden in all lymphoid tissues had increased and approached levels observed in terminal disease, yet there was no evidence of nervous system invasion. These results indicate the first site of CWD prion entry is in the oropharynx, and the initial phase of prion amplification occurs in the oropharyngeal lymphoid tissues followed by rapid dissemination to systemic lymphoid tissues. This lymphoid replication phase appears to precede neuroinvasion.IMPORTANCE Chronic wasting disease (CWD) is a universally fatal transmissible spongiform encephalopathy affecting cervids, and natural infection occurs through oral and nasal mucosal exposure to infectious prions. Terminal disease is characterized by PrPCWD accumulation in the brain and lymphoid tissues of affected animals. However, the initial sites of prion accumulation and pathways of prion spread during early CWD infection remain unknown. To investigate the chronological events of early prion pathogenesis, we exposed deer to CWD prions and monitored the tissue distribution of PrPCWD over the first 4 months of infection. We show CWD uptake occurs in the oropharynx with initial prion replication in the draining oropharyngeal lymphoid tissues, rapidly followed by dissemination to systemic lymphoid tissues without evidence of neuroinvasion. These data highlight the two phases of CWD infection: a robust prion amplification in systemic lymphoid tissues prior to neuroinvasion and establishment of a carrier state.

In utero transmission and tissue distribution of chronic wasting disease-associated prions in free-ranging Rocky Mountain elk.

The presence of disease-associated prions in tissues and bodily fluids of chronic wasting disease (CWD)-infected cervids has received much investigation, yet little is known about mother-to-offspring transmission of CWD. Our previous work demonstrated that mother-to-offspring transmission is efficient in an experimental setting. To address the question of relevance in a naturally exposed free-ranging population, we assessed maternal and fetal tissues derived from 19 elk dam-calf pairs collected from free-ranging Rocky Mountain elk from north-central Colorado, a known CWD endemic region. Conventional immunohistochemistry identified three of 19 CWD-positive dams, whereas a more sensitive assay [serial protein misfolding cyclic amplification (sPMCA)] detected CWD prion seeding activity (PrPCWD) in 15 of 19 dams. PrPCWD distribution in tissues was widespread, and included the central nervous system (CNS), lymphoreticular system, and reproductive, secretory, excretory and adipose tissues. Interestingly, five of 15 sPMCA-positive dams showed no evidence of PrPCWD in either CNS or lymphoreticular system, sites typically assessed in diagnosing CWD. Analysis of fetal tissues harvested from the 15 sPMCA-positive dams revealed PrPCWD in 80 % of fetuses (12 of 15), regardless of gestational stage. These findings demonstrated that PrPCWD is more abundant in peripheral tissues of CWD-exposed elk than current diagnostic methods suggest, and that transmission of prions from mother to offspring may contribute to the efficient transmission of CWD in naturally exposed cervid populations.

Complement protein C3 exacerbates prion disease in a mouse model of chronic wasting disease.

Accumulating evidence shows a critical role of the complement system in facilitating attachment of prions to both B cells and follicular dendritic cells and assisting in prion replication. Complement activation intensifies disease in prion-infected animals, and elimination of complement components inhibits prion accumulation, replication and pathogenesis. Chronic wasting disease (CWD) is a highly infectious prion disease of captive and free-ranging cervid populations that utilizes the complement system for efficient peripheral prion replication and most likely efficient horizontal transmission. Here we show that complete genetic or transient pharmacological depletion of C3 prolongs incubation times and significantly delays splenic accumulation in a CWD transgenic mouse model. Using a semi-quantitative prion amplification scoring system we show that C3 impacts disease progression in the early stages of disease by slowing the rate of prion accumulation and/or replication. The delayed kinetics in prion replication correlate with delayed disease kinetics in mice deficient in C3. Taken together, these data support a critical role of C3 in peripheral CWD prion pathogenesis.

Genetic depletion of complement receptors CD21/35 prevents terminal prion disease in a mouse model of chronic wasting disease.

The complement system has been shown to facilitate peripheral prion pathogenesis. Mice lacking complement receptors CD21/35 partially resist terminal prion disease when infected i.p. with mouse-adapted scrapie prions. Chronic wasting disease (CWD) is an emerging prion disease of captive and free-ranging cervid populations that, similar to scrapie, has been shown to involve the immune system, which probably contributes to their relatively facile horizontal and environmental transmission. In this study, we show that mice overexpressing the cervid prion protein and susceptible to CWD (Tg(cerPrP)5037 mice) but lack CD21/35 expression completely resist clinical CWD upon peripheral infection. CD21/35-deficient Tg5037 mice exhibit greatly impaired splenic prion accumulation and replication throughout disease, similar to CD21/35-deficient murine prion protein mice infected with mouse scrapie. TgA5037;CD21/35(-/-) mice exhibited little or no neuropathology and deposition of misfolded, protease-resistant prion protein associated with CWD. CD21/35 translocate to lipid rafts and mediates a strong germinal center response to prion infection that we propose provides the optimal environment for prion accumulation and replication. We further propose a potential role for CD21/35 in selecting prion quasi-species present in prion strains that may exhibit differential zoonotic potential compared with the parental strains.

The Role of Glial Cells in Neurobiology and Prion Neuropathology.

Prion diseases are rare and neurodegenerative diseases that are characterized by the misfolding and infectious spread of the prion protein in the brain, causing progressive and irreversible neuronal loss and associated clinical and behavioral manifestations in humans and animals, ultimately leading to death. The brain has a complex network of neurons and glial cells whose crosstalk is critical for function and homeostasis. Although it is established that prion infection of neurons is necessary for clinical disease to occur, debate remains in the field as to the role played by glial cells, namely astrocytes and microglia, and whether these cells are beneficial to the host or further accelerate disease. Here, we review the current literature assessing the complex morphologies of astrocytes and microglia, and the crosstalk between these two cell types, in the prion-infected brain.

Adipose-Derived Mesenchymal Stromal Cells Co-Cultured with Primary Mixed Glia to Reduce Prion-Induced Inflammation.

Mesenchymal stromal cells (MSCs) are potent regulators of inflammation through the production of anti-inflammatory cytokines, chemokines, and growth factors. These cells show an ability to regulate neuroinflammation in the context of neurodegenerative diseases such as prion disease and other protein misfolding disorders. Prion diseases can be sporadic, acquired, or genetic; they can result from the misfolding and aggregation of the prion protein in the brain. These diseases are invariably fatal, with no available treatments. One of the earliest signs of disease is the activation of astrocytes and microglia and associated inflammation, which occurs prior to detectable prion aggregation and neuronal loss; thus, the anti-inflammatory and regulatory properties of MSCs can be harvested to treat astrogliosis in prion disease. Recently, we showed that adipose-derived MSCs (AdMSCs) co-cultured with BV2 cells or primary mixed glia reduce prion-induced inflammation through paracrine signaling. This paper describes a reliable treatment using stimulated AdMSCs to decrease prion-induced inflammation. A heterozygous population of AdMSCs can easily be isolated from murine adipose tissue and expanded in culture. Stimulating these cells with inflammatory cytokines enhances their ability to both migrate toward prion-infected brain homogenate and produce anti-inflammatory modulators in response. Together, these techniques can be used to investigate the therapeutic potential of MSCs on prion infection and can be adapted for other protein misfolding and neuroinflammatory diseases.

Intranasally delivered mesenchymal stromal cells decrease glial inflammation early in prion disease.

Mesenchymal stromal cells (MSCs) are an intriguing avenue for the treatment of neurological disorders due to their ability to migrate to sites of neuroinflammation and respond to paracrine signaling in those sites by secreting cytokines, growth factors, and other neuromodulators. We potentiated this ability by stimulating MSCs with inflammatory molecules, improving their migratory and secretory properties. We investigated the use of intranasally delivered adipose-derived MSCs (AdMSCs) in combating prion disease in a mouse model. Prion disease is a rare, lethal neurodegenerative disease that results from the misfolding and aggregation of the prion protein. Early signs of this disease include neuroinflammation, activation of microglia, and development of reactive astrocytes. Later stages of disease include development of vacuoles, neuronal loss, abundant aggregated prions, and astrogliosis. We demonstrate the ability of AdMSCs to upregulate anti-inflammatory genes and growth factors when stimulated with tumor necrosis factor alpha (TNFα) or prion-infected brain homogenates. We stimulated AdMSCs with TNFα and performed biweekly intranasal deliveries of AdMSCs on mice that had been intracranially inoculated with mouse-adapted prions. At early stages in disease, animals treated with AdMSCs showed decreased vacuolization throughout the brain. Expression of genes associated with Nuclear Factor-kappa B (NF-κB) and Nod-Like Receptor family pyrin domain containing 3 (NLRP3) inflammasome signaling were decreased in the hippocampus. AdMSC treatment promoted a quiescent state in hippocampal microglia by inducing changes in both number and morphology. Animals that received AdMSCs showed a decrease in both overall and reactive astrocyte number, and morphological changes indicative of homeostatic astrocytes. Although this treatment did not prolong survival or rescue neurons, it demonstrates the benefits of MSCs in combatting neuroinflammation and astrogliosis.

A multiplexed, paired-pooled droplet digital PCR assay for detection of SARS-CoV-2 in saliva.

In response to the SARS-CoV-2 pandemic, we developed a multiplexed, paired-pool droplet digital PCR (MP4) screening assay. Key features of our assay are the use of minimally processed saliva, 8-sample paired pools, and reverse-transcription droplet digital PCR (RT-ddPCR) targeting the SARS-CoV-2 nucleocapsid gene. The limit of detection was determined to be 2 and 12 copies per µl for individual and pooled samples, respectively. Using the MP4 assay, we routinely processed over 1,000 samples a day with a 24-h turnaround time and over the course of 17 months, screened over 250,000 saliva samples. Modeling studies showed that the efficiency of 8-sample pools was reduced with increased viral prevalence and that this could be mitigated by using 4-sample pools. We also present a strategy for, and modeling data supporting, the creation of a third paired pool as an additional strategy to employ under high viral prevalence.

Sensitivity of protein misfolding cyclic amplification versus immunohistochemistry in ante-mortem detection of chronic wasting disease.

As the only prion disease affecting free-ranging animals, ante-mortem identification of affected cervids has become paramount in understanding chronic wasting disease (CWD) pathogenesis, prevalence and control of horizontal or vertical transmission. To seek maximal sensitivity in ante-mortem detection of CWD infection, this study used paired tonsil biopsy samples collected at various time points from 48 CWD-exposed cervids to compare blinded serial protein misfolding cyclic amplification (sPMCA) with the assay long considered the 'gold standard' for CWD detection, immunohistochemistry (IHC). sPMCA-negative controls (34 % of the samples evaluated) included tissues from mock-inoculated animals and unspiked negative controls, all of which tested negative throughout the course of the study. It was found that sPMCA on tonsil biopsies detected CWD infection significantly earlier (2.78 months, 95 % confidence interval 2.40-3.15) than conventional IHC. Interestingly, a correlation was observed between early detection by sPMCA and host PRNP genotype. These findings demonstrate that in vitro-amplification assays provide enhanced sensitivity and advanced detection of CWD infection in the peripheral tissues of cervids, with a potential role for spike or substrate genotype in sPMCA amplification efficiency.

Detection of chronic wasting disease prions in salivary, urinary, and intestinal tissues of deer: potential mechanisms of prion shedding and transmission.

Efficient horizontal transmission is a signature trait of chronic wasting disease (CWD) in cervids. Infectious prions shed into excreta appear to play a key role in this facile transmission, as has been demonstrated by bioassays of cervid and transgenic species and serial protein misfolding cyclic amplification (sPMCA). However, the source(s) of infectious prions in these body fluids has yet to be identified. In the present study, we analyzed tissues proximate to saliva, urine, and fecal production by sPMCA in an attempt to elucidate this unique aspect of CWD pathogenesis. Oropharyngeal, urogenital, and gastrointestinal tissues along with blood and obex from CWD-exposed cervids (comprising 27 animals and >350 individual samples) were analyzed and scored based on the apparent relative CWD burden. PrP(CWD)-generating activity was detected in a range of tissues and was highest in the salivary gland, urinary bladder, and distal intestinal tract. In the same assays, blood from the same animals and unseeded normal brain homogenate controls (n = 116 of 117) remained negative. The PrP-converting activity in peripheral tissues varied from 10(-11)- to 10(0)-fold of that found in brain of the same animal. Deer with highest levels of PrP(CWD) amplification in the brain had higher and more widely disseminated prion amplification in excretory tissues. Interestingly, PrP(CWD) was not demonstrable in these excretory tissues by conventional Western blotting, suggesting a low prion burden or the presence of protease-sensitive infectious prions destroyed by harsh proteolytic treatments. These findings offer unique insights into the transmission of CWD in particular and prion infection and trafficking overall.

Adipose-derived mesenchymal stromal cells decrease prion-induced glial inflammation in vitro.

Prion diseases are characterized by the cellular prion protein, PrPC, misfolding and aggregating into the infectious prion protein, PrPSc, which leads to neurodegeneration and death. An early sign of disease is inflammation in the brain and the shift of resting glial cells to reactive astrocytes and activated microglia. Few therapeutics target this stage of disease. Mesenchymal stromal cells produce anti-inflammatory molecules when exposed to inflammatory signals and damaged tissue. Here, we show that adipose-derived mesenchymal stromal cells (AdMSCs) migrate toward prion-infected brain homogenate and produce the anti-inflammatory molecules transforming growth factor ß (TGFß) and tumor necrosis factor-stimulated gene 6 (TSG-6). In an in vitro model of prion exposure of both primary mixed glia and BV2 microglial cell line, co-culturing with AdMSCs led to a significant decrease in inflammatory cytokine mRNA and markers of reactive astrocytes and activated microglia. This protection against in vitro prion-associated inflammatory responses is independent of PrPSc replication. These data support a role for AdMSCs as a beneficial therapeutic for decreasing the early onset of glial inflammation and reprogramming glial cells to a protective phenotype.

Investigating transmission of SARS-CoV-2 using novel face mask sampling: a protocol for an observational prospective study of index cases and their contacts in a congregate setting.

INTRODUCTION: This study aims to measure how transmission of SARS-CoV-2 occurs in communities and to identify conditions that lend to increased transmission focusing on congregate situations. We will measure SARS-CoV-2 in exhaled breath of asymptomatic and symptomatic persons using face mask sampling-a non-invasive method for SARS-CoV-2 detection in exhaled air. We aim to detect transmission clusters and identify risk factors for SARS-CoV-2 transmission in presymptomatic, asymptomatic and symptomatic individuals. METHODS AND ANALYSIS: In this observational prospective study with daily follow-up, index cases and their respective contacts are identified at each participating institution. Contact definitions are based on Centers for Disease Control and Prevention and local health department guidelines. Participants will wear masks with polyvinyl alcohol test strips adhered to the inside for 2 hours daily. The strips are applied to all masks used over at least 7 days. In addition, self-administered nasal swabs and (optional) finger prick blood samples are performed by participants. Samples are tested by standard PCR protocols and by novel antigen tests. ETHICS AND DISSEMINATION: This study was approved by the Colorado Multiple Institutional Review Board and the WHO Ethics Review Committee. From the data generated, we will analyse transmission clusters and risk factors for transmission of SARS-CoV-2 in congregate settings. The kinetics of asymptomatic transmission and the evaluation of non-invasive tools for detection of transmissibility are of crucial importance for the development of more targeted control interventions-and ultimately to assist with keeping congregate settings open that are essential for our social fabric. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov (#NCT05145803).

siRNA Therapeutics for Protein Misfolding Diseases of the Central Nervous System.

Nanoparticles have been used to deliver siRNA to tissues and cells to silence specific genes in diverse organisms. Research and clinical application of nanoparticles like liposomes for drug delivery requires targeting them to specific anatomic regions or cell types, while avoiding off-target effects or clearance by the liver, kidney, or the immune system. Delivery to the central nervous system (CNS) presents additional challenges to cross the blood-brain barrier (BBB) to specific cell types like neurons, astrocytes, or glia. Here, we describe the generation of three different liposomal siRNA delivery vehicles to the CNS using the thin film hydration method. Utilizing cationic or anionic liposomes protects the siRNA from serum nucleases and proteases en route. To deliver the siRNA specifically to the CNS, the liposomes are complexed to a peptide that acts as a neuronal address by binding to nicotinic acetylcholine receptors (nAchRs). When injected intravenously or instilled intranasally, these liposome-siRNA-peptide complexes (LSPCs) or peptide addressed liposome-encapsulated therapeutic siRNA (PALETS) resist serum degradation, effectively cross the BBB, and deliver siRNA to AchR-expressing cells to suppress protein expression in the CNS.

CCR2+Ly-6Chi monocytes are crucial for the effector phase of autoimmunity in the central nervous system.

The chemokine receptor CCR2 plays a vital role for the induction of autoimmunity in the central nervous system. However, it remains unclear how the pathogenic response is mediated by CCR2-bearing cells. By combining bone marrow chimerism with gene targeting we detected a mild disease-modulating role of CCR2 during experimental autoimmune encephalomyelitis, a model for central nervous system autoimmunity, on radio-resistant cells that was independent from targeted CCR2 expression on endothelia. Interestingly, absence of CCR2 on lymphocytes did not influence autoimmune demyelination. In contrast, engagement of CCR2 on accessory cells was required for experimental autoimmune encephalomyelitis induction. CCR2+Ly-6Chi monocytes were rapidly recruited to the inflamed central nervous system and were crucial for the effector phase of disease. Selective depletion of this specific monocyte subpopulation through engagement of CCR2 strongly reduced central nervous system autoimmunity. Collectively, these data indicate a disease-promoting role of CCR2+Ly-6Chi monocytes during autoimmune inflammation of the central nervous system.

Perturbation of T-cell development by insertional mutation of a PrP transgene.

The normal cellular form of the prion protein PrP(C) is a glycosylphosphatidylinositol-linked cell-surface glycoprotein expressed primarily by cells of the nervous and immune systems. There is evidence to suggest that PrP(C) is involved in cell signalling and cellular homeostasis. We have investigated the immune composition of peripheral lymphoid tissue in PrP-/-, wild-type, tg19 and tga20 strains of mice, which express 0, 1-, 3-5- and 4-7-fold higher levels of PrP(C), respectively, relative to wild-type mice. Our data show that tga20 mice have a reduced number of spleen T-cell receptor (TCR)-alphabeta(+) T cells and an increased number of TCR-gammadelta(+) T cells compared with wild-type mice. This was not seen in tg19 mice, which also express elevated levels of PrP(C). In addition, we have found that the Prnp transgene in the tga20 genome is located centrally on chromosome 17, in or around genes involved in T-cell development. Significantly, mRNA transcripts from pre-TCR-alpha (pTalpha), a T-cell development gene located on mouse chromosome 17, are drastically reduced in tga20 mice, indicative of a perturbation in pTalpha gene regulation. We propose that the immune cell phenotype of tga20 mice may be caused by the insertional mutation of the Prnp transgene into the pTalpha gene or its regulatory elements.

Lipopeptide Delivery of siRNA to the Central Nervous System.

RNA interference is a relatively new tool used to silence specific genes in diverse biological systems. The development of this promising new technique for research and therapeutic use in studying and treating neurological diseases has been hampered by the lack of an efficient way to deliver siRNA transvascularly across the blood-brain barrier (BBB) to the central nervous system (CNS). Here we describe the generation of three different liposomal siRNA delivery vehicles to the CNS using the thin film hydration method. Utilizing cationic or anionic liposomes protects the siRNA from serum nucleases and proteases en route. To deliver the siRNA specifically to the CNS, the liposomes are complexed to a peptide that acts as a neuronal address by binding to nicotinic acetylcholine receptors (nAchRs). When injected intravenously, these liposome-siRNA-peptide complexes (LSPCs) or peptide addressed liposome encapsulated therapeutic siRNA (PALETS) resist serum degradation, effectively cross the BBB and deliver siRNA to AchR-expressing cells to suppress protein expression in the CNS.