Broad-Spectrum and Personalized Guide RNAs for CRISPR/Cas9 HIV-1 Therapeutics.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 system has been used to excise the HIV-1 proviral genome from latently infected cells, potentially offering a cure for HIV-infected patients. Recent studies have shown that most published HIV-1 guide RNAs (gRNAs) do not account for the diverse viral quasispecies within or among patients, which continue to diversify with time even in long-term antiretroviral therapy (ART)-suppressed patients. Given this observation, proviral genomes were deep sequenced from 23 HIV-1-infected patients in the Drexel Medicine CNS AIDS Research and Eradication Study cohort at two different visits. Based on the spectrum of integrated proviral DNA polymorphisms observed, three gRNA design strategies were explored: based on the patient's own HIV-1 sequences (personalized), based on consensus sequences from a large sample of patients [broad-spectrum (BS)], or a combination of both approaches. Using a bioinformatic algorithm, the personalized gRNA design was predicted to cut 46 of 48 patient samples at 90% efficiency, whereas the top 4 BS gRNAs (BS4) were predicted to excise provirus from 44 of 48 patient samples with 90% efficiency. Using a mixed design with the top three BS gRNAs plus one personalized gRNA (BS3 + PS1) resulted in predicted excision of provirus from 45 of 48 patient samples with 90% efficiency. In summary, these studies used an algorithmic design strategy to identify potential BS gRNAs to target a spectrum of HIV-1 long teriminal repeat (LTR) quasispecies for use with a small HIV-1-infected population. This approach should advance CRISPR/Cas9 excision technology taking into account the extensive molecular heterogeneity of HIV-1 that persists in situ after prolonged ART.

Specific amino acids in HIV-1 Vpr are significantly associated with differences in patient neurocognitive status.

Even in the era of combination antiretroviral therapies used to combat human immunodeficiency virus type 1 (HIV-1) infection, up to 50 % of well-suppressed HIV-1-infected patients are still diagnosed with mild neurological deficits referred to as HIV-associated neurocognitive disorders (HAND). The multifactorial nature of HAND likely involves the HIV-1 accessory protein viral protein R (Vpr) as an agent of neuropathogenesis. To investigate the effect of naturally occurring variations in Vpr on HAND in well-suppressed HIV-1-infected patients, bioinformatic analyses were used to correlate peripheral blood-derived Vpr sequences with patient neurocognitive performance, as measured by comprehensive neuropsychological assessment and the resulting Global Deficit Score (GDS). Our studies revealed unique associations between GDS and the presence of specific amino acid changes in peripheral blood-derived Vpr sequences [neuropsychological impairment Vpr (niVpr) variants]. Amino acids N41 and A55 in the Vpr sequence were associated with more pronounced neurocognitive deficits (higher GDS). In contrast, amino acids I37 and S41 were connected to measurably lower GDS. All niVpr variants were also detected in DNA isolated from HIV-1-infected brain tissues. The implication of these results is that niVpr variants alter the genesis and/or progression of HAND through differences in Vpr-mediated effects in the peripheral blood and/or the brain.

Mitochondrial Haplogroup Influences Motor Function in Long-Term HIV-1-Infected Individuals.

Evolutionary divergence of the mitochondrial genome has given rise to distinct haplogroups. These haplogroups have arisen in specific geographical locations and are responsible for subtle functional changes in the mitochondria that may provide an evolutionary advantage in a given environment. Based on these functional differences, haplogroups could define disease susceptibility in chronic settings. In this study, we undertook a detailed neuropsychological analysis of a cohort of long-term HIV-1-infected individuals in conjunction with sequencing of their mitochondrial genomes. Stepwise regression analysis showed that the best model for predicting both working memory and declarative memory were age and years since diagnosis. In contrast, years since diagnosis and sub-haplogroup were significantly predictive of psychomotor speed. Consistent with this, patients with haplogroup L3e obtained better scores on psychomotor speed and dexterity tasks when compared to the remainder of the cohort, suggesting that this haplogroup provides a protective advantage when faced with the combined stress of HIV-1 infection and long-term antiretroviral therapies. Differential performance on declarative memory tasks was noted for individuals with other sub-L haplogroups, but these differences were not as robust as the association between L3e and psychomotor speed and dexterity tasks. This work provides evidence that mitochondrial haplogroup is related to neuropsychological test performance among patients in chronic disease settings such as HIV-1 infection.

Utilization of HIV-1 envelope V3 to identify X4- and R5-specific Tat and LTR sequence signatures.

BACKGROUND: HIV-1 entry is a receptor-mediated process directed by the interaction of the viral envelope with the host cell CD4 molecule and one of two co-receptors, CCR5 or CXCR4. The amino acid sequence of the third variable (V3) loop of the HIV-1 envelope is highly predictive of co-receptor utilization preference during entry, and machine learning predictive algorithms have been developed to characterize sequences as CCR5-utilizing (R5) or CXCR4-utilizing (X4). It was hypothesized that while the V3 loop is predominantly responsible for determining co-receptor binding, additional components of the HIV-1 genome may contribute to overall viral tropism and display sequence signatures associated with co-receptor utilization. RESULTS: The accessory protein Tat and the HlV-1 long terminal repeat (LTR) were analyzed with respect to genetic diversity and compared by Jensen-Shannon divergence which resulted in a correlation with both mean genetic diversity as well as the absolute difference in genetic diversity between R5- and X4-genome specific trends. As expected, the V3 domain of the gp120 protein was enriched with statistically divergent positions. Statistically divergent positions were also identified in Tat amino acid sequences within the transactivation and TAR-binding domains, and in nucleotide positions throughout the LTR. We further analyzed LTR sequences for putative transcription factor binding sites using the JASPAR transcription factor binding profile database and found several putative differences in transcription factor binding sites between R5 and X4 HIV-1 genomes, specifically identifying the C/EBP sites I and II, and Sp site III to differ with respect to sequence configuration for R5 and X4 LTRs. CONCLUSION: These observations support the hypothesis that co-receptor utilization coincides with specific genetic signatures in HIV-1 Tat and the LTR, likely due to differing transcriptional regulatory mechanisms and selective pressures applied within specific cellular targets during the course of productive HIV-1 infection.

HIV-1 Genetic Variation Resulting in the Development of New Quasispecies Continues to Be Encountered in the Peripheral Blood of Well-Suppressed Patients.

As a result of antiretroviral therapeutic strategies, human immunodeficiency virus type 1 (HIV-1) infection has become a long-term clinically manageable chronic disease for many infected individuals. However, despite this progress in therapeutic control, including undetectable viral loads and CD4+ T-cell counts in the normal range, viral mutations continue to accumulate in the peripheral blood compartment over time, indicating either low level reactivation and/or replication. Using patients from the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort, whom have been sampled longitudinally for more than 7 years, genetic change was modeled against to the dominant integrated proviral quasispecies with respect to selection pressures such as therapeutic interventions, AIDS defining illnesses, and other factors. Phylogenetic methods based on the sequences of the LTR and tat exon 1 of the HIV-1 proviral DNA quasispecies were used to obtain an estimate of an average mutation rate of 5.3 nucleotides (nt)/kilobasepair (kb)/year (yr) prior to initiation of antiretroviral therapy (ART). Following ART the baseline mutation rate was reduced to an average of 1.02 nt/kb/yr. The post-ART baseline rate of genetic change, however, appears to be unique for each patient. These studies represent our initial steps in quantifying rates of genetic change among HIV-1 quasispecies using longitudinally sampled sequences from patients at different stages of disease both before and after initiation of combination ART. Notably, while long-term ART reduced the estimated mutation rates in the vast majority of patients studied, there was still measurable HIV-1 mutation even in patients with no detectable virus by standard quantitative assays. Determining the factors that affect HIV-1 mutation rates in the peripheral blood may lead to elucidation of the mechanisms associated with changes in HIV-1 disease severity.

Defining differential genetic signatures in CXCR4- and the CCR5-utilizing HIV-1 co-linear sequences.

The adaptation of human immunodeficiency virus type-1 (HIV-1) to an array of physiologic niches is advantaged by the plasticity of the viral genome, encoded proteins, and promoter. CXCR4-utilizing (X4) viruses preferentially, but not universally, infect CD4+ T cells, generating high levels of virus within activated HIV-1-infected T cells that can be detected in regional lymph nodes and peripheral blood. By comparison, the CCR5-utilizing (R5) viruses have a greater preference for cells of the monocyte-macrophage lineage; however, while R5 viruses also display a propensity to enter and replicate in T cells, they infect a smaller percentage of CD4+ T cells in comparison to X4 viruses. Additionally, R5 viruses have been associated with viral transmission and CNS disease and are also more prevalent during HIV-1 disease. Specific adaptive changes associated with X4 and R5 viruses were identified in co-linear viral sequences beyond the Env-V3. The in silico position-specific scoring matrix (PSSM) algorithm was used to define distinct groups of X4 and R5 sequences based solely on sequences in Env-V3. Bioinformatic tools were used to identify genetic signatures involving specific protein domains or long terminal repeat (LTR) transcription factor sites within co-linear viral protein R (Vpr), trans-activator of transcription (Tat), or LTR sequences that were preferentially associated with X4 or R5 Env-V3 sequences. A number of differential amino acid and nucleotide changes were identified across the co-linear Vpr, Tat, and LTR sequences, suggesting the presence of specific genetic signatures that preferentially associate with X4 or R5 viruses. Investigation of the genetic relatedness between X4 and R5 viruses utilizing phylogenetic analyses of complete sequences could not be used to definitively and uniquely identify groups of R5 or X4 sequences; in contrast, differences in the genetic diversities between X4 and R5 were readily identified within these co-linear sequences in HIV-1-infected patients.

Somatic cell-like features of cloned mouse embryos prepared with cultured myoblast nuclei.

Cloning by somatic cell nuclear transfer requires silencing of the donor cell gene expression program and the initiation of the embryonic gene expression program (nuclear reprogramming). Failure to silence the donor cell program could lead to altered embryonic phenotypes. Cloned mouse embryos produced using myoblast nuclei fail to thrive in standard embryo culture media but flourish in somatic cell culture media favored by the donor myoblasts themselves, forming blastocysts at a significant rate, with robust morphologies, high total cell number, and a normal allocation of cells to the inner cell mass in most embryos. Myoblast cloned embryos continue expressing the GLUT4 glucose transporter, which is typically expressed in muscle but not in preimplantation stage embryos. Myoblast clones also exhibit precocious enrichment of GLUT1 at the cell surface. Both myoblast and cumulus cell cloned embryos exhibit enhanced rates of glucose uptake. These observations indicate that silencing of the donor cell genome during cloning either is incomplete or occurs progressively over the course of preimplantation development. As a result, cloned embryos initially exhibit many somatic cell-like characteristics. Tetraploid constructs, which possess a transplanted somatic cell genome plus the oocyte-derived chromosomes, exhibit a more embryonic-like pattern of gene expression and culture preference. We conclude that preimplantation stage cloned embryos have profoundly altered characteristics that are donor cell type specific and that exposure of cloned embryos to standard embryo culture conditions may lead to disruptions in basic homeostasis and inhibition of a range of essential processes including further nuclear reprogramming, contributing to cloned embryo demise.

Trophectoderm-specific expression of the X-linked Bex1/Rex3 gene in preimplantation stage mouse embryos.

The Bex1/Rex3 gene was recently identified as an X-linked gene that is differentially expressed between parthenogenetic and normal fertilized, preimplantation stage mouse embryos. The Bex1/Rex3 gene appears to be expressed preferentially from the maternal X chromosome in blastocysts, but from either X chromosome in later stage embryonic tissues and adult tissues. To investigate whether differential expression of the Bex1/Rex3 gene between normal and parthenogenetic blastocyst stage embryos reflects genomic imprinting at the Bex1/Rex3 locus itself, or instead is the result of preferential inactivation of the paternal X chromosome or differences in timing of cellular differentiation, we examined in detail the expression pattern of the Bex1/Rex3 mRNA in normal preimplantation stage embryos, and compared its expression between androgenetic, gynogenetic, and normal fertilized embryos. Expression data reveal that the Bex1/Rex3 gene is initially transcribed at the 2-cell stage, transiently induced at the 8-cell stage, and then increases in expression again at the blastocyst stage. Very little expression is observed in isolated inner cell masses, indicating selective expression in the trophectoderm. Comparisons of Bex1/Rex3 mRNA expression between male and female androgenetic and control embryos and gynogenetic embros failed to reveal any significant difference in expression between the different classes of embryos at the 8-cell stage, or the expanding blastocyst stage (121 hr post-hCG). At the late blastocyst stage (141 hr post-hCG), expression was significantly lower in XY control embryos as compared with XX controls. Bex1/Rex3 mRNA expression did not differ between XX and XY androgenones at the blastocyst stage or between gynogenones and XX control embryos. Thus, the Bex1/Rex3 gene does not appear to be regulated directly by genomic imprinting during the preimplantation period, just as it is not regulated by imprinting at later stages. Apparent differences in gene expression may arise through the effects of trophectoderm-specific expression coupled with differences in timing of trophectoderm differentiation between the different classes of embryos and effects of preferential paternal X chromosome inactivation (XCI).