Site-specific gene addition can allow stable transgene expression for gene therapy. When possible, this is preferred over the use of promiscuously integrating vectors, which are sometimes associated with clonal expansion1 and oncogenesis2. Site-specific endonucleases that can induce high rates of targeted genome editing are finding increasing applications in biological discovery and gene therapy3. However, two safety concerns persist: (1) endonuclease-associated adverse effects, both on4 and off-target5,6; and (2) oncogene activation caused by promoter integration, even without nucleases7. Here, we perform recombinant adeno-associated virus (rAAV) mediated promoterless gene targeting without nucleases and demonstrate amelioration of the bleeding diathesis in haemophilia B mice. In particular, we target a promoterless human coagulation factor IX (hF9) gene to the liver-expressed albumin (Alb) locus. hF9 is targeted, along with a preceding 2A-peptide coding sequence, to be integrated just upstream to the Alb stop codon. While hF9 is fused to Alb at the DNA and RNA levels, two separate proteins are synthesized by way of ribosomal skipping. Thus, hF9 expression is linked to robust hepatic albumin expression without disrupting it. We injected an AAV8-hF9 vector into neonatal and adult mice and achieved on-target integration into ~0.5% of the albumin alleles in hepatocytes. We established that hF9 was produced only from on-target integration, and ribosomal skipping was highly efficient. Stable hF9 plasma levels at 7–20% of normal were obtained, and treated factor IX deficient mice had normal coagulation times. In conclusion, transgene integration as a 2A-fusion to a highly expressed endogenous gene may obviate the requirement for nucleases and/or vector-borne promoters. This method may allow for safe and efficacious gene targeting in both infants and adults by greatly diminishing off-target effects while still providing therapeutic levels of expression from integration.

Systemic delivery of mRNA-based therapeutics remains a challenging issue for preclinical and clinical studies. Here, we describe new lipid-like nanoparticles (TT-LLNs) developed through an orthogonal array design, which demonstrates improved delivery efficiency of mRNA encoding luciferase in vitro by over 350-fold with significantly reduced experimental workload. One optimized TT3 LLN, termed O-TT3 LLNs, was able to restore the human factor IX (hFIX) level to normal physiological values in FIX-knockout mice. Consequently, these mRNA based nanomaterials merit further development for therapeutic applications.

The myelodysplastic syndromes (MDS) comprise a heterogenous group of myeloid disorders with a highly variable disease course. Diagnostic criteria to better stratify patients with MDS continue to evolve, based on morphology, cytogenetics, and the presence of cytopenias. More accurate classification of patients will allow for better treatment guidance. Treatment encompasses supportive care, treatment of anemia, low-intensity therapy, and high-intensity therapy. This portion of the guidelines focuses on diagnostic classification, molecular abnormalities, therapeutic options, and recommended treatment approaches.

Globin genes are subject to tissue-specific and developmental stage-specific regulation. A switch from human fetal ('y)-to adult (,B)-globin expression occurs within erythroid precursor cells of the adult lineage. Previously we and others showed by targeted gene disruption that the zinc finger gene, erythroid Kruippel-like factor (EKLF), is required for expression of the f3-globin gene in mice, presumably through interaction with a high-affinity binding site in the proximal promoter. To examine the role of EKLF in the developmental regulation of the human y-globin gene we interbred EKLF heterozygotes (+/-) with mice harboring a human ,B-globin yeast artificial chromosome transgene. We find that in the absence of EKLF, while human f-globin expression is dramatically reduced, 'y-globin transcripts are elevated -5-fold. Impaired silencing of y-globin expression identifies EKLF as the first transcription factor participating quantitatively in the y-globin to f8-globin switch. Our findings are compatible with a competitive model of switching in which EKLF mediates an adult stage-specific interaction between the f3-globin gene promoter and the locus control region that excludes the y-globin gene.

Systemic gene transfer provides new opportunities for the analysis of gene function and gene regulation in vivo, as well as for human gene therapy. We used the chloramphenicol acetyltransferase reporter gene to examine several parameters important for the development of efficient, cationic liposome-mediated, intravenous (IV) gene transfer in mice. We then demonstrated that this approach can produce high level expression of biologically important genes. Specifically, we assessed the relationship of expression vector design to the level of systemic gene expression produced, and compared transfection levels produced by intravenously injecting DNA alone versus DNA⅐liposome complexes. We found that both the position of the heterologous intron, and the promoter element used in the expression plasmid, significantly affected the level of systemic gene expression produced. Although intravenous injection of plasmid DNA alone transfected every tissue analyzed, liposome-mediated delivery was much more efficient. We also established that repeated IV injection of DNA⅐liposome complexes produced high level systemic transfection. The second injection of DNA⅐liposome complexes produced levels of gene expression at least as high as those following a single IV injection. Thus, unlike some viral vectors, a neutralizing host-immune response does not limit re-expression, following reinjection of DNA⅐liposome complexes.Finally, we showed that the expression vectors which produced the highest levels of chloramphenicol acetyltransferase reporter gene expression could also produce high level expression of two colony stimulating factor genes in mice. Specifically, IV injection of liposomes complexed to expression vectors into which we had inserted either the murine granulocyte-macrophagecolony stimulating factor cDNA or the human granulocyte-CSF cDNA, produced circulating levels of the corresponding colony stimulating factor gene product comparable to levels which have been shown previously to be both biologically and therapeutically significant.