Glycopeptide characterization of Sf9-derived SARS-CoV-2 spike protein recombinant vaccine candidates expedited by the use of glycopeptide libraries.

RATIONALE: We report the N-glycosylation pattern of Sf9 insect cell-derived recombinant spike proteins being developed as candidate vaccine antigens for SARS-CoV-2 (COVID-19) (Sanofi). The method has been optimised to produce peptides with single, isolated glycosylation sites using multiple protease digests. The development and use of glycopeptide libraries from previous developmental phases allowed for faster analysis than processing datasets from individual batches from first principles. METHODS: Purified spike proteins were reduced, alkylated, and digested with proteolytic enzymes. Three different protease digests were utilised to generate peptides with isolated glycosylation sites. The glycopeptides were then analysed using a Waters Q-TOF while using a data-dependent acquisition mass spectrometry experiment. Glycopeptide mapping data processing and glycan classification were performed using Genedata Expressionist via a specialised workflow that used libraries of previously detected glycopeptides to greatly reduce processing time. RESULTS: Two different spike proteins from six manufacturers were analysed. There was a strong similarity at each site across batches and manufacturers. The majority of the glycans present were of the truncated class, although at sites N61, N234, and N717/714 high mannose structures were dominant and at N1173/1170 aglycosylation was dominant for both variant proteins. A comparison was performed on a commercially available spike protein and our results were found to be similar to those of earlier reports. CONCLUSIONS: Our data clearly show that the overall glycosylation pattern of both spike protein variants was highly similar from batch to batch, and between materials produced at different manufacturing facilities. The use of our glycopeptide libraries greatly expedited the generation of site-specific glycan occupancy data for a large glycoprotein. We compared our method with previously obtained data from a commercially available insect cell-derived spike protein and the results were comparable to published findings.

Biomedical research in the Cloud: considerations for researchers and organizations moving to (or adding) cloud computing resources.

As biomedical research data grow, researchers need reliable and scalable solutions for storage and compute. There is also a need to build systems that encourage and support collaboration and data sharing, to result in greater reproducibility. This has led many researchers and organizations to use cloud computing [1]. The cloud not only enables scalable, on-demand resources for storage and compute, but also collaboration and continuity during virtual work, and can provide superior security and compliance features. Moving to or adding cloud resources, however, is not trivial or without cost, and may not be the best choice in every scenario. The goal of this workshop is to explore the benefits of using the cloud in biomedical and computational research, and considerations (pros and cons) for a range of scenarios including individual researchers, collaborative research teams, consortia research programs, and large biomedical research agencies / organizations.

The association of biomarkers with pain and function in acute and subacute low back pain: a secondary analysis of an RCT.

BACKGROUND: Low back pain (LBP) is a common musculoskeletal condition and a major cause of disability worldwide. Previous studies have found associations of biomarkers with pain and pain-related disability in LBP patients. This study aimed to explore the association between serum biomarkers and pain and disability in patients with acute or subacute axial LBP. METHODS: This study was ancillary to a parent randomized controlled trial. Enrolled participants were randomized into three intervention groups: one of two types of spinal manipulation or medical care. In the parent study, 107 adults who experienced a new episode of LBP within 3 months prior to enrollment were recruited. For this study, 90 of these 107 participants consented to have blood samples obtained, which were drawn immediately before the beginning of treatment. Seven biomarkers were chosen based on previous literature and analyzed. Clinical outcomes were pain and Oswestry Disability Index (ODI) evaluated at baseline and 4 weeks. Spearman's |r| was used to study the association of initial levels of each biomarker with pain and ODI scores at baseline and with changes in outcome scores from baseline to 4 weeks (end of treatment) within each intervention group. RESULTS: At baseline, 4 of 7 biomarkers had an association with pain that was |r| ≥ .20: neuropeptide Y (NPY) (r = 0.23, p = .028), E-Selectin (r = 0.22, p = .043), vitamin D ((r = - 0.32, p = .002), and c-reactive protein (CRP) (r = 0.37, p = .001). No baseline biomarker had an association with disability that was |r| ≥ 0.20. For the correlations of baseline biomarkers with 4-week change in outcomes, vitamin D showed a correlation with change in disability and/or pain (|r| ≥ 0.20, p > .05) in manipulation-related groups, while CRP, NPY, and E-selectin along with TNFα, Substance P and RANTES showed at least one correlation with change in pain or disability (|r| ≥ 0.20, p > .05) in at least one of the treatment groups. CONCLUSIONS: In 90 LBP patients, the analyzed biomarkers, especially vitamin D, represent a small set of potential candidates for further research aimed at individualizing patient care. Overall, the associations investigated in the current study are an initial step in identifying the direct mechanisms of LBP and predicting outcomes of manipulation-related treatments or medical care. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01211613, Date of Registration: September 29, 2010, https://clinicaltrials.gov/ct2/show/NCT01211613?term=schneider&cond=Low+Back+Pain&cntry=US&state=US%3APA&draw=2&rank=1.

Attribute Analytics Performance Metrics from the MAM Consortium Interlaboratory Study.

The multi-attribute method (MAM) was conceived as a single assay to potentially replace multiple single-attribute assays that have long been used in process development and quality control (QC) for protein therapeutics. MAM is rooted in traditional peptide mapping methods; it leverages mass spectrometry (MS) detection for confident identification and quantitation of many types of protein attributes that may be targeted for monitoring. While MAM has been widely explored across the industry, it has yet to gain a strong foothold within QC laboratories as a replacement method for established orthogonal platforms. Members of the MAM consortium recently undertook an interlaboratory study to evaluate the industry-wide status of MAM. Here we present the results of this study as they pertain to the targeted attribute analytics component of MAM, including investigation into the sources of variability between laboratories and comparison of MAM data to orthogonal methods. These results are made available with an eye toward aiding the community in further optimizing the method to enable its more frequent use in the QC environment.

Advancing Structure Characterization of PS-80 by Charge-Reduced Mass Spectrometry and Software-Assisted Composition Analysis.

The commercially available Polysorbate 80 (PS-80) is a highly heterogeneous product. It is a complex and structurally diverse mixture consisting of polymeric species containing polyoxyethylenes (POEs), fatty acid esters, with/or without a carbohydrate core. The core is primarily sorbitan, with some isosorbide and sorbitol. Depending on the sources of fatty acids and the degrees of esterification, multiple combinations of fatty acid esters are commonly observed. A number of POE intermediates, such as polyoxyethylene glycols, POE-sorbitans, POE-isosorbides, and an array of fatty acid esters from these intermediates remain in the raw material as well. The complex composition of PS-80 is difficult to control and poses a significant characterization challenge for its use in the pharmaceutical industry. Here, we present a novel solution for PS-80 characterization using ultra high-performance liquid chromatography coupled with charge-reduction high resolution mass spectrometry. Post column co-infusion of triethylamine focused the signal into mainly singly charged molecular ions and reduced the extent of in-source fragmentation, resulting in a simpler ion map and enhanced measurement of PS-80 species. The data processing workflow is designed to programmatically identify PS-80 component classes and reduce the burden of manually analyzing complex MS data. The 2-dimensional graphical representation of the data helps visualize these features. Together, these innovative methodologies enabled us to analyze components in PS-80 with unprecedented detail and shall be a useful tool to study formulation and stability of pharmaceutical preparations. The power of this approach was demonstrated by comparing the composition of PS-80 obtained from different vendors.

New Peak Detection Performance Metrics from the MAM Consortium Interlaboratory Study.

The Multi-Attribute Method (MAM) Consortium was initially formed as a venue to harmonize best practices, share experiences, and generate innovative methodologies to facilitate widespread integration of the MAM platform, which is an emerging ultra-high-performance liquid chromatography-mass spectrometry application. Successful implementation of MAM as a purity-indicating assay requires new peak detection (NPD) of potential process- and/or product-related impurities. The NPD interlaboratory study described herein was carried out by the MAM Consortium to report on the industry-wide performance of NPD using predigested samples of the NISTmAb Reference Material 8671. Results from 28 participating laboratories show that the NPD parameters being utilized across the industry are representative of high-resolution MS performance capabilities. Certain elements of NPD, including common sources of variability in the number of new peaks detected, that are critical to the performance of the purity function of MAM were identified in this study and are reported here as a means to further refine the methodology and accelerate adoption into manufacturer-specific protein therapeutic product life cycles.

Unusual fragmentation of derivatized cysteine-containing peptides.

RATIONALE: Modification of cysteines by aminoethylation results in side chains similar to those of lysine. Trypsin cleaves at this modified residue and this labeling method can facilitate the analysis of proteins, specifically antibodies. In this work, the ability to identify peptides containing aminoethylated cysteines is investigated through digestion, covalent labeling, and low-energy ion fragmentation. METHODS: A prototype antibody was reduced, aminoethylated, and digested with either Lys-N or Glu-C. The resulting peptides were amidinated with SMTA and analyzed by PSD in a MALDI-TOF/TOF mass spectrometer or by CID in an ESI ion trap/orbitrap mass spectrometer. RESULTS: PSD and CID fragmentation of peptides with an amidinated aminoethylated cysteine can produce an intense characteristic loss from this modified residue. A neutral loss of 118 Da or charged loss of 119 Da is observed when peptides have low charges. This fragment can form when the cysteine is located in any position in the peptide. The rationalization for this ion is that the amidino group can be initially neutral or protonated and initiates fragmentation. CONCLUSIONS: The combination of a dual-labeling technique and low-energy fragmentation produces an abundant diagnostic ion for the analysis of cysteine-containing peptides. These 118 and 119 Da losses are observed when protons are sequestered.

Nephele: a cloud platform for simplified, standardized and reproducible microbiome data analysis.

Motivation: Widespread interest in the study of the microbiome has resulted in data proliferation and the development of powerful computational tools. However, many scientific researchers lack the time, training, or infrastructure to work with large datasets or to install and use command line tools. Results: The National Institute of Allergy and Infectious Diseases (NIAID) has created Nephele, a cloud-based microbiome data analysis platform with standardized pipelines and a simple web interface for transforming raw data into biological insights. Nephele integrates common microbiome analysis tools as well as valuable reference datasets like the healthy human subjects cohort of the Human Microbiome Project (HMP). Nephele is built on the Amazon Web Services cloud, which provides centralized and automated storage and compute capacity, thereby reducing the burden on researchers and their institutions. Availability and implementation: https://nephele.niaid.nih.gov and https://github.com/niaid/Nephele. Contact: darrell.hurt@nih.gov.

Use of Cysteine Aminoethylation To Identify the Hypervariable Peptides of an Antibody.

Aminoethylation of cysteines can provide enzymatically cleavable sites. The ability to obtain peptides containing antibody complementarity determining regions (CDRs) with aminoethylated cysteines was investigated. Because cysteines are often located N-terminal to CDRs, digestion with Lys-N enables acquisition of peptides with CDRs. Lys-N peptides containing an aminoethylated cysteine at the N-terminus were also amidinated. Subsequent collisional activation yields a unique loss of 118 Da that originates from this modified residue, providing a signature ion for cysteine-containing peptides. The relative cleavage efficiencies for Lys-N and trypsin are also compared.

A Novel Triethylphosphonium Charge Tag on Peptides: Synthesis, Derivatization, and Fragmentation.

Charge tagging is a peptide derivatization process that commonly localizes a positive charge on the N-terminus. Upon low energy activation (e.g., collision-induced dissociation or post-source decay) of charge tagged peptides, relatively few fragment ions are produced due to the absence of mobile protons. In contrast, high energy fragmentation, such as 157 nm photodissociation, typically leads to a series of a-type ions. Disadvantages of existing charge tags are that they can produce mobile protons or that they are undesirably large and bulky. Here, we investigate a small triethylphosphonium charge tag with two different linkages: amide (158 Da) and amidine bonds (157 Da). Activation of peptides labeled with a triethylphosphonium charge tag through an amide bond can lead to loss of the charge tag and the production of protonated peptides. This enables low intensity fragment ions from both the protonated and charge tagged peptides to be observed. Triethylphosphonium charge tagged peptides linked through an amidine bond are more stable. Post-source decay and photodissociation yield product ions that primarily contain the charge tag. Certain amidine induced fragments are also observed. The previously reported tris(trimethoxyphenyl) phosphonium acetic acid N-hydroxysuccinimidyl ester charge tag shows a similar fragment ion distribution, but the mass of the triethylphosphonium tag label is 415 Da smaller. Graphical Abstract ᅟ.

Distinguishing Aspartic and Isoaspartic Acids in Peptides by Several Mass Spectrometric Fragmentation Methods.

Six ion fragmentation techniques that can distinguish aspartic acid from its isomer, isoaspartic acid, were compared. MALDI post-source decay (PSD), MALDI 157 nm photodissociation, tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) charge tagging in PSD and photodissociation, ESI collision-induced dissociation (CID), electron transfer dissociation (ETD), and free-radical initiated peptide sequencing (FRIPS) with CID were applied to peptides containing either aspartic or isoaspartic acid. Diagnostic ions, such as the y-46 and b+H2O, are present in PSD, photodissociation, and charge tagging. c•+57 and z-57 ions are observed in ETD and FRIPS experiments. For some molecules, aspartic and isoaspartic acid yield ion fragments with significantly different intensities. ETD and charge tagging appear to be most effective at distinguishing these residues. Graphical Abstract ᅟ.

Factors Affecting the Production of Aromatic Immonium Ions in MALDI 157 nm Photodissociation Studies.

Immonium ions are commonly observed in the high energy fragmentation of peptide ions. In a MALDI-TOF/TOF mass spectrometer, singly charged peptides photofragmented with 157 nm VUV light yield a copious abundance of immonium ions, especially those from aromatic residues. However, their intensities may vary from one peptide to another. In this work, the effect of varying amino acid position, peptide length, and peptide composition on immonium ion yield is investigated. Internal immonium ions are found to have the strongest intensity, whereas immonium ions arising from C-terminal residues are the weakest. Peptide length and competition among residues also strongly influence the immonium ion production. Quantum calculations provide insights about immonium ion structures and the fragment ion conformations that promote or inhibit immonium ion formation.

The NIH 3D Print Exchange: A Public Resource for Bioscientific and Biomedical 3D Prints.

The National Institutes of Health (NIH) has launched the NIH 3D Print Exchange, an online portal for discovering and creating bioscientifically relevant 3D models suitable for 3D printing, to provide both researchers and educators with a trusted source to discover accurate and informative models. There are a number of online resources for 3D prints, but there is a paucity of scientific models, and the expertise required to generate and validate such models remains a barrier. The NIH 3D Print Exchange fills this gap by providing novel, web-based tools that empower users with the ability to create ready-to-print 3D files from molecular structure data, microscopy image stacks, and computed tomography scan data. The NIH 3D Print Exchange facilitates open data sharing in a community-driven environment, and also includes various interactive features, as well as information and tutorials on 3D modeling software. As the first government-sponsored website dedicated to 3D printing, the NIH 3D Print Exchange is an important step forward to bringing 3D printing to the mainstream for scientific research and education.

PEGylated poly(ethylene imine) copolymer-delivered siRNA inhibits HIV replication in vitro.

RNA interference is increasingly being utilized for the specific targeting and down-regulation of disease-causing genes, including targeting viral infections such as HIV. T lymphocytes, the primary target for HIV, are very difficult to treat with gene therapy applications such as RNA interference because of issues with drug delivery. To circumvent these problems, we investigated poly(ethylene imine) (PEI) as a method of improving transfection efficiency of siRNA to T lymphocytes. Additionally, polyethylene glycol (PEG) moieties were engrafted to the PEI polymers with the goals of improving stability and reducing cytotoxicity. Initial studies on PEG-PEI/siRNA polyplex formation, size and their interaction with cell membranes demonstrated their feasibility as drug delivery agents. Assays with lymphocytes revealed low cytotoxicity profiles of the polyplexes at pharmacologically relevant concentrations with PEGylated copolymers obtaining the best results. Successful transfection of a T cell line or primary T cells with siRNA was observed via flow cytometry and confocal microscopy. Finally, the biological effect of copolymer-delivered siRNA was measured. Of particular significance, siRNA targeted to the HIV gene nef and delivered by one of the PEG-PEI copolymers in repetitive treatments every 2-3 days was observed to inhibit HIV replication to the same extent as azidothymidine over the course of 15 days.

Carbosilane dendrimers to transfect human astrocytes with small interfering RNA targeting human immunodeficiency virus.

BACKGROUND: HIV infection of the CNS is the principle cause of HIV-associated dementia in adults and encephalopathy in children. Gene therapy techniques such as small interfering RNA (siRNA) possess great potential in drug development, but first they must overcome the key obstacle of reaching the interior of the affected cells. A successful delivery vector for anti-HIV drugs that is capable of crossing the blood-brain barrier (BBB) could provide a way of addressing this issue. Non-viral vectors such as dendrimers offer a means for effectively delivering and transfecting siRNA to the target cells. OBJECTIVE: To evaluate the application of gene therapy for reducing HIV replication in human astrocytes. METHODS: We used the 2G-NN16 amino-terminated carbosilane dendrimer as a method for delivering siRNA to HIV-infected human astrocytes. We tested the cytotoxicity in human astrocytoma cells caused by 2G-NN16 and dendriplexes formed with siRNA (siRNA/2G-NN16) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide (MTT) and lactate dehydrogenase assays. The ability to transfect human astrocytes with siRNA/2G-NN16 dendriplexes was tested by flow cytometry and immunofluorescence microscopy. To assess the potential capability of siRNA/2G-NN16 dendriplexes for crossing the BBB, we used an in vitro transcytosis assay with bovine brain microvascular endothelial cells. HIV-1 inhibition assays using 2G-NN16 and siRNA/2G-NN16 dendriplexes were determined by quantification of the viral load from culture supernatants of the astrocytes. RESULTS: A gradual time-controlled degradation of the 2G-NN16 dendrimer and liberation of its siRNA cargo between 12 and 24 hours was observed via gel electrophoresis. There was no cytotoxicity in HIV-infected or non-infected human astrocytoma cells when treated with up to 24 microg/mL of 2G-NN16 dendrimer or siRNA/2G-NN16 dendriplexes, and siRNA/2G-NN16 dendriplexes were seen to successfully transfect human astrocytes even after crossing an in vitro BBB model. More interestingly, transfected siRNA was observed to exert a biologic effect, as dendriplexes were shown to down-regulate the housekeeping gene GAPDH and to reduce replication of HIV-1 strains X4-HIV NL4-3 and R5-HIV BaL in human astrocytes. CONCLUSIONS: The 2G-NN16 dendrimer successfully delivers and transfects siRNA to HIV-infected human astrocytes and achieves gene silencing without causing cytotoxicity.

Gene therapy in HIV-infected cells to decrease viral impact by using an alternative delivery method.

The ability of dendrimer 2G-[Si{O(CH(2))(2)N(Me)(2) (+)(CH(2))(2)NMe(3) (+)(I(-))(2)}](8) (NN16) to transfect a wide range of cell types, as well as the possible biomedical application in direct or indirect inhibition of HIV replication, was investigated. Cells implicated in HIV infection such as primary peripheral blood mononuclear cells (PBMC) and immortalized suspension cells (lymphocytes), primary macrophages and dendritic cells, and immortalized adherent cells (astrocytes and trophoblasts) were analyzed. Dendrimer toxicity was evaluated by mitochondrial activity, cell membrane rupture, release of lactate dehydrogenase, erythrocyte hemolysis, and the effect on global gene expression profiles using whole-genome human microarrays. Cellular uptake of genetic material was determined using flow cytometry and confocal microscopy. Transfection efficiency and gene knockdown was investigated using dendrimer-delivered antisense oligonucleotides and small interfering RNA (siRNA). Very little cytotoxicity was detected in a variety of cells relevant to HIV infection and erythrocytes after NN16 dendrimer treatment. Imaging of cellular uptake showed high transfection efficiency of genetic material in all cells tested. Interestingly, NN16 further enhanced the reduction of HIV protein 24 antigen release by antisense oligonucleotides due to improved transfection efficiency. Finally, the dendrimer complexed with siRNA exhibited therapeutic potential by specifically inhibiting cyclooxygenase-2 gene expression in HIV-infected nervous system cells. NN16 dendrimers demonstrated the ability to transfect genetic material into a vast array of cells relevant to HIV pathology, combining high efficacy with low toxicity. These results suggest that NN16 dendrimers have the potential to be used as a versatile non-viral vector for gene therapy against HIV infection.

Associations among hip and shoulder range of motion and shoulder injury in professional baseball players.

CONTEXT: The overhead throwing motion is complex, and restrictions in range of motion (ROM) at the hip may place additional demands on the shoulder that lead to injury. However, the relationship between hip and shoulder ROM in athletes with and without a history of shoulder injury is unknown. OBJECTIVE: To (1) determine if differences exist in hip and shoulder ROM between professional baseball players with a history of shoulder injury and those with no history of shoulder injury and (2) assess relationships between hip and shoulder ROM in these players. DESIGN: Cross-sectional study. PATIENTS OR OTHER PARTICIPANTS: Fifty-seven professional baseball players. MAIN OUTCOME MEASURE(S): Outcome measures consisted of hip extension and internal rotation, shoulder internal and external rotation, glenohumeral internal-rotation deficit, and history of shoulder injury. Differences in shoulder and hip ROM were assessed with a 1-way analysis of variance. Associations between hip and shoulder ROM were assessed with linear regression. RESULTS: Nonpitchers with a history of shoulder injury had more external rotation and less internal rotation of the shoulder than nonpitchers with no history of shoulder injury. Glenohumeral internal-rotation deficit was greater in both pitchers and nonpitchers with a history of shoulder injury. The relationship between dominant hip extension and shoulder external rotation was significant for pitchers with a history of shoulder injury and nonpitchers with a history of shoulder injury. CONCLUSIONS: Shoulder injury may be associated with specific measures of hip and shoulder ROM, and hip extension and shoulder external rotation may be related in baseball players with a history of shoulder injury. Additional research is necessary to understand the specific mechanisms of shoulder injury in the throwing athlete.

Mobilization of endogenous retroviruses in mice after infection with an exogenous retrovirus.

Mammalian genomes harbor a large number of retroviral elements acquired as germ line insertions during evolution. Although many of the endogenous retroviruses are defective, several contain one or more intact viral genes that are expressed under certain physiological or pathological conditions. This is true of the endogenous polytropic retroviruses that generate recombinant polytropic murine leukemia viruses (MuLVs). In these recombinants the env gene sequences of exogenous ecotropic MuLVs are replaced with env gene sequences from an endogenous polytropic retrovirus. Although replication-competent endogenous polytropic retroviruses have not been observed, the recombinant polytropic viruses are capable of replicating in numerous species. Recombination occurs during reverse transcription of a virion RNA heterodimer comprised of an RNA transcript from an endogenous polytropic virus and an RNA transcript from an exogenous ecotropic MuLV RNA. It is possible that homodimers corresponding to two full-length endogenous RNA genomes are also packaged. Thus, infection by an exogenous virus may result not only in recombination with endogenous sequences, but also in the mobilization of complete endogenous retrovirus genomes via pseudotyping within exogenous retroviral virions. We report that the infection of mice with an ecotropic virus results in pseudotyping of intact endogenous viruses that have not undergone recombination. The endogenous retroviruses infect and are integrated into target cell genomes and subsequently replicate and spread as pseudotyped viruses. The mobilization of endogenous retroviruses upon infection with an exogenous retrovirus may represent a major interaction of exogenous retroviruses with endogenous retroviruses and may have profound effects on the pathogenicity of retroviral infections.

Characterization of carbosilane dendrimers as effective carriers of siRNA to HIV-infected lymphocytes.

One of the primary limitations of RNA interference as a technique for gene regulation is effective delivery of siRNA into the target cells. Dendrimers are nanoparticles that are increasingly being used as oligonucleotide and drug delivery vehicles. We have developed amino-terminated carbosilane dendrimers (CBS) as a means to protect and transport siRNA. Initially, stability studies showed that CBS bind siRNA via electrostatic interactions. Dendrimer-bound siRNA was found to be resistant to degradation by RNase. Cytotoxicity assays of CBS/siRNA dendriplexes with peripheral blood mononuclear cells (PBMC) and the lymphocytic cell line SupT1 revealed a maximum safe dendrimer concentration of 25 microg/ml. Next, utilizing flow cytometry and confocal microscopy, lymphocytes were seen to be successfully transfected by fluorochrome-labeled siRNA either naked or complexed with CBS. Dendriplexes with +/- charge ratio of 2 were determined to have the highest transfection efficiency while maintaining a low level of toxicity in these systems including hard-to-transfect HIV-infected PBMC. Finally, CBS/siRNA dendriplexes were shown to silence GAPDH expression and reduce HIV replication in SupT1 and PBMC. These results point to the possibility of utilizing dendrimers such as CBS to deliver and transfect siRNA into lymphocytes thus allowing the use of RNA interference as a potential alternative therapy for HIV infection.