Expression of a Targeted λ1 Light Chain Gene Is Developmentally Regulated and Independent of Igκ Rearrangements

Immunoglobulin light chain (IgL) rearrangements occur more frequently at Igκ than at Igλ. Previous results suggested that the unrearranged Igκ locus negatively regulates Igλ transcription and/or rearrangement. Here, we demonstrate that expression of a VJλ1-joint inserted into its physiological position in the Igλ locus is independent of Igκ rearrangements. Expression of the inserted VJλ1 gene segment is developmentally controlled like that of a VJκ-joint inserted into the Igκ locus and furthermore coincides developmentally with the occurrence of Igκ rearrangements in wild-type mice. We conclude that developmentally controlled transcription of a gene rearrangement in the Igλ locus occurs in the presence of an unrearranged Igκ locus and is therefore not negatively regulated by the latter. Our data also indicate light chain editing in ∼30% of λ1 expressing B cell progenitors

Direct in vivo V(H) to J(H) rearrangement violating the 12/23 rule

V(D)J recombination at the immunoglobulin heavy chain (IgH) locus follows the 12/23 rule to ensure the correct assembly of the variable region gene segments. Here, we report characterization of an in vivo model that allowed us to study recombination violating the 12/23 rule, namely a mouse strain lacking canonical D elements in its IgH locus. We demonstrate that V(H) to J(H) joining can support the generation of all B cell subsets. However, the process is inefficient in that B cells and antibodies derived from the D(H)-less allele are not detectable if the latter is combined with a wild-type IgH allele. There is no preferential usage of any particular V(H) gene family or J(H) element in V(H)J(H) junctions, indicating that 23/23-guided recombination is possible, but is a low frequency event at the IgH locus in vivo

The Roles of Individual Mammalian Argonautes in RNA Interference In Vivo

Argonaute 2 (Ago2) is the only mammalian Ago protein capable of mRNA cleavage. It has been reported that the activity of the short interfering RNA targeting coding sequence (CDS), but not 3′ untranslated region (3′UTR) of an mRNA, is solely dependent on Ago2 in vitro. These studies utilized extremely high doses of siRNAs and overexpressed Ago proteins, as well as were directed at various highly expressed reporter transgenes. Here we report the effect of Ago2 in vivo on targeted knockdown of several endogenous genes by siRNAs, targeting both CDS and 3′UTR. We show that siRNAs targeting CDS lose their activity in the absence of Ago2, whereas both Ago1 and Ago3 proteins contribute to residual 3′UTR-targeted siRNA-mediated knockdown observed in the absence of Ago2 in mouse liver. Our results provide mechanistic insight into two components mediating RNAi under physiological conditions: mRNA cleavage dependent and independent. In addition our results contribute a novel consideration for designing most efficacious siRNA molecules with the preference given to 3′UTR targeting as to harness the activity of several Ago proteins.Alnylam Pharmaceuticals (Firm

Rearrangement and Expression of Immunoglobulin Light Chain Genes Can Precede Heavy Chain Expression during Normal B Cell Development in Mice

In mouse mutants incapable of expressing μ chains, VκJκ joints are detected in the CD43+ B cell progenitors. In agreement with these earlier results, we show by a molecular single cell analysis that 4–7% of CD43+ B cell progenitors in wild-type mice rearrange immunoglobulin (Ig)κ genes before the assembly of a productive VHDHJH joint. Thus, μ chain expression is not a prerequisite to Igκ light chain gene rearrangements in normal development. Overall, ∼15% of the total CD43+ B cell progenitor population carry Igκ gene rearrangements in wild-type mice. Together with the results obtained in the mouse mutants, these data fit a model in which CD43+ progenitors rearrange IgH and Igκ loci independently, with a seven times higher frequency in the former. In addition, we show that in B cell progenitors VκJκ joining rapidly initiates κ chain expression, irrespective of the presence of a μ chain

Highly Effective Gene Transfection In Vivo by Alkylated Polyethylenimine

We mechanistically explored the effect of increased hydrophobicity of the polycation on the efficacy and specificity of gene delivery in mice. N-Alkylated linear PEIs with varying alkyl chain lengths and extent of substitution were synthesized and characterized by biophysical methods. Their in vivo transfection efficiency, specificity, and biodistribution were investigated. N-Ethylation improves the in vivo efficacy of gene expression in the mouse lung 26-fold relative to the parent polycation and more than quadruples the ratio of expression in the lung to that in all other organs. N-Propyl-PEI was the best performer in the liver and heart (581- and 3.5-fold enhancements, resp.) while N-octyl-PEI improved expression in the kidneys over the parent polymer 221-fold. As these enhancements in gene expression occur without changing the plasmid biodistribution, alkylation does not alter the cellular uptake but rather enhances transfection subsequent to cellular uptake.National Institutes of Health (U.S.) (NIH Grant 2-R37-EB000244-29)National Science Foundation (U.S.) (NSF-0070319 )National Institutes of Health (U.S.) (NIH GM68762

Lipidoid Nanoparticles Containing PD-L1 siRNA Delivered In Vivo Enter Kupffer Cells and Enhance NK and CD8+ T Cell-mediated Hepatic Antiviral Immunity

Effective clinical application of antiviral immunotherapies necessitates enhancing the functional state of natural killer (NK) and CD8+ T cells. An important mechanism for the establishment of viral persistence in the liver is the activation of the PD-1/PD-L1 inhibitory pathway. To examine the role of hepatic myeloid PD-L1 expression during viral infection, we determined the magnitude and quality of antiviral immune responses by administering PD-L1 short-interfering RNA (siRNA) encapsulated in lipidoid nanoparticles (LNP) in mice. Our studies indicate that Kupffer cells (KC) preferentially engulfed PD-L1 LNP within a short period of time and silenced Pdl1 during adenovirus and MCMV infection leading to enhanced NK and CD8+ T cell intrahepatic accumulation, effector function (interferon (IFN)-γ and granzyme B (GrB) production), CD8+ T cell–mediated viral clearance, and memory. Our results demonstrate that PD-L1 knockdown on KCs is central in determining the outcome of liver viral infections, and they represent a new class of gene therapy