The loop position of shRNAs and pre-miRNAs is critical for the accuracy of dicer processing in vivo.

Short hairpin RNA (shRNA)-induced RNAi is used for biological discovery and therapeutics. Dicer, whose normal role is to liberate endogenous miRNAs from their precursors, processes shRNAs into different biologically active siRNAs, affecting their efficacy and potential for off-targeting. We found that, in cells, Dicer induced imprecise cleavage events around the expected sites based on the previously described 5'/3' counting rules. These promiscuous noncanonical cleavages were abrogated when the cleavage site was positioned 2 nt from a bulge or loop. Interestingly, we observed that the ~1/3 of mammalian endogenous pre-miRNAs that contained such structures were more precisely processed by Dicer. Implementing a "loop-counting rule," we designed potent anti-HCV shRNAs with substantially reduced off-target effects. Our results suggest that Dicer recognizes the loop/bulge structure in addition to the ends of shRNAs/pre-miRNAs for accurate processing. This has important implications for both miRNA processing and future design of shRNAs for RNAi-based genetic screens and therapies.

Correlation of antigen expression with epigenetic modifications after rAAV delivery of a human factor IX variant in mice and rhesus macaques.

We investigated long-term human coagulation factor IX (huFIX) expression of a novel variant when delivered into mice and rhesus macaques and compared transduction efficiencies using two different adeno-associated virus (AAV) capsids. In hemophilic mice injected with KP1-packaged recombinant AAV (rAAV) expressing the hyperactive FIX variant specific activity plasma levels were 10-fold or 2-fold enhanced when compared with wild-type or Padua huFIX injected mice, respectively. In rhesus macaques AAV-LK03 capsid outperformed AAV-KP1 in terms of antigen expression and liver transduction. Two animals from each group showed sustained low-level huFIX expression at 3 months after administration, while one animal from each group lost huFIX mRNA and protein expression over time, despite comparable vector copies. We investigated whether epigenetic differences in the vector episomes could explain this loss of transcription. Cut&Tag analysis revealed lower levels of activating histone marks in the two animals that lost expression. When comparing rAAV genome associated histone modifications in rhesus macaques with those in mice injected with the same vector, the activating histone marks were starkly decreased in macaque-derived episomes. Differential epigenetic marking of AAV genomes may explain different expression profiles in mice and rhesus macaques, as well as the wide dose response variation observed in primates in both preclinical and human clinical trials.

Pediatric Vascular Surgery: A Review of Cases from a Dedicated Pediatric Vascular Surgery Clinic.

BACKGROUND: The experience in pediatric vascular diseases is limited in the United Kingdom and worldwide due to their rarity and variations in practice. We looked at types of cases presenting to a dedicated pediatric vascular clinic. METHODS: Medical records of children seen in a dedicated pediatric vascular clinic at a tertiary referral service between 2016 and 2022 were reviewed. These patients were either seen for the first time in that clinic or had their appointments as a follow-up after inpatient review or intervention while being under the care of pediatric teams in local hospitals. RESULTS: Fifty-five patients (34 males) were seen aged between 4 months and 17 years (mean 9.5 years). Common presentations were limb length discrepancy secondary to iatrogenic arterial occlusion, follow-up after bypass for trauma, lower limb swelling or discoloration, and varicose veins. Operative procedures included lower limb bypass, angioplasty, ligation of aneurysms, and varicose vein surgery. CONCLUSIONS: Pediatric vascular conditions are uncommon and therefore most vascular surgeons and trainees will have little exposure to such cases. Intervention is needed for arterial injury secondary to penetrating or iatrogenic trauma. A national registry is required for these rare cases to gain prospective data that can help build up more evidence for educational purposes and to establish guidelines.

The 3'tsRNAs are aminoacylated: Implications for their biogenesis.

Emerging evidence indicates that tRNA-derived small RNAs (tsRNAs) are involved in fine-tuning gene expression and become dysregulated in various cancers. We recently showed that the 22nt LeuCAG3´tsRNA from the 3´ end of tRNALeu is required for efficient translation of a ribosomal protein mRNA and ribosome biogenesis. Inactivation of this 3´tsRNA induced apoptosis in rapidly dividing cells and suppressed the growth of a patient-derived orthotopic hepatocellular carcinoma in mice. The mechanism involved in the generation of the 3´tsRNAs remains elusive and it is unclear if the 3´-ends of 3´tsRNAs are aminoacylated. Here we report an enzymatic method utilizing exonuclease T to determine the 3´charging status of tRNAs and tsRNAs. Our results showed that the LeuCAG3´tsRNA, and two other 3´tsRNAs are fully aminoacylated. When the leucyl-tRNA synthetase (LARS1) was inhibited, there was no change in the total tRNALeu concentration but a reduction in both the charged tRNALeu and LeuCAG3´tsRNA, suggesting the 3´tsRNAs are fully charged and originated solely from the charged mature tRNA. Altering LARS1 expression or the expression of various tRNALeu mutants were also shown to affect the generation of the LeuCAG3´tsRNA further suggesting they are created in a highly regulated process. The fact that the 3´tsRNAs are aminoacylated and their production is regulated provides additional insights into their importance in post-transcriptional gene regulation that includes coordinating the production of the protein synthetic machinery.

Evolution of a Human-Specific Tandem Repeat Associated with ALS.

Tandem repeats are proposed to contribute to human-specific traits, and more than 40 tandem repeat expansions are known to cause neurological disease. Here, we characterize a human-specific 69 bp variable number tandem repeat (VNTR) in the last intron of WDR7, which exhibits striking variability in both copy number and nucleotide composition, as revealed by long-read sequencing. In addition, greater repeat copy number is significantly enriched in three independent cohorts of individuals with sporadic amyotrophic lateral sclerosis (ALS). Each unit of the repeat forms a stem-loop structure with the potential to produce microRNAs, and the repeat RNA can aggregate when expressed in cells. We leveraged its remarkable sequence variability to align the repeat in 288 samples and uncover its mechanism of expansion. We found that the repeat expands in the 3'-5' direction, in groups of repeat units divisible by two. The expansion patterns we observed were consistent with duplication events, and a replication error called template switching. We also observed that the VNTR is expanded in both Denisovan and Neanderthal genomes but is fixed at one copy or fewer in non-human primates. Evaluating the repeat in 1000 Genomes Project samples reveals that some repeat segments are solely present or absent in certain geographic populations. The large size of the repeat unit in this VNTR, along with our multiplexed sequencing strategy, provides an unprecedented opportunity to study mechanisms of repeat expansion, and a framework for evaluating the roles of VNTRs in human evolution and disease.

Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals.

Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.

Outcome of Femoral Angioplasty/Stenting Procedures in Different Ethnic Groups in England: A Retrospective Analysis of Hospital Episode Statistics and Review of Literature.

PURPOSE: Various studies, mainly from North America, report worse outcomes in ethnic minority populations submitted to revascularization for peripheral arterial disease (PAD). Limited nationwide data in relation to ethnicity are available from Europe. OBJECTIVE: The objective of the study is to compare the outcomes of femoral angioplasty/stenting procedures among different ethnic groups in England during the 10-year period from 2006 to 2015. MATERIALS AND METHODS: The "Hospital Episode Statistics" database has been searched using International Classification of Diseases, Tenth Revision (ICD-10) codes to identify all cases of femoral angioplasty or stenting from English NHS Hospitals between January 1, 2006, and December 31, 2015. Subsequent mortality, second open or endovascular infrainguinal procedures, and major amputations on the same side within 2 years after the first procedure have been recorded. Patients were broadly categorized according to ethnicity as whites, Asians, and blacks. Chi-square test was used to demonstrate significant differences among ethnic groups and odds ratios (ORs) were calculated using white ethnic group as reference. RESULTS: A total number of 70 887 femoral endovascular procedures were recorded in patients from the 3 ethnic groups. Two-year mortality in whites, Asians, and blacks was 18.3%, 22.1%, and 19.5% (p<0.001); rates of second endovascular procedure were 12.1%, 13.1%, and 13.5% (p=0.24); rates of open infrainguinal procedure were 5.6%, 4.5%, and 8.0% (p<0.001); and rates of major amputation were 4.8%, 4.1%, and 7.0% (p<0.001), respectively. Mortality was higher in Asians (OR=1.26, 95% confidence interval [CI]=1.10-1.45, p<0.01) compared with whites. On the contrary, blacks underwent more open arterial operations (OR=1.48, 95% CI=1.19-1.83, p<0.01) and more amputations (OR=1.49, 95% CI=1.18-1.87, p<0.01). There were no significant differences in the rates of second endovascular procedures. CONCLUSION: Two-year mortality after femoral angioplasty/stenting is higher in Asians, whereas risk of limb loss is higher in blacks compared with whites. Reasons of these ethnic differences in outcomes following femoral endovascular procedures for PAD merit further study.

A transfer-RNA-derived small RNA regulates ribosome biogenesis.

Transfer-RNA-derived small RNAs (tsRNAs; also called tRNA-derived fragments) are an abundant class of small non-coding RNAs whose biological roles are not well understood. Here we show that inhibition of a specific tsRNA, LeuCAG3'tsRNA, induces apoptosis in rapidly dividing cells in vitro and in a patient-derived orthotopic hepatocellular carcinoma model in mice. This tsRNA binds at least two ribosomal protein mRNAs (RPS28 and RPS15) to enhance their translation. A decrease in translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits. These data establish a post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer.

Evaluating the state of the science for adeno-associated virus integration: An integrated perspective.

On August 18, 2021, the American Society of Gene and Cell Therapy (ASGCT) hosted a virtual roundtable on adeno-associated virus (AAV) integration, featuring leading experts in preclinical and clinical AAV gene therapy, to further contextualize and understand this phenomenon. Recombinant AAV (rAAV) vectors are used to develop therapies for many conditions given their ability to transduce multiple cell types, resulting in long-term expression of transgenes. Although most rAAV DNA typically remains episomal, some rAAV DNA becomes integrated into genomic DNA at a low frequency, and rAAV insertional mutagenesis has been shown to lead to tumorigenesis in neonatal mice. Currently, the risk of rAAV-mediated oncogenesis in humans is theoretical because no confirmed genotoxic events have been reported to date. However, because insertional mutagenesis has been reported in a small number of murine studies, there is a need to characterize this genotoxicity to inform research, regulatory needs, and patient care. The purpose of this white paper is to review the evidence of rAAV-related host genome integration in animal models and possible risks of insertional mutagenesis in patients. In addition, technical considerations, regulatory guidance, and bioethics are discussed.

Promoterless, Nuclease-Free Genome Editing Confers a Growth Advantage for Corrected Hepatocytes in Mice With Methylmalonic Acidemia.

BACKGROUND AND AIMS: Adeno-associated viral (AAV) gene therapy has shown great promise as an alternative treatment for metabolic disorders managed using liver transplantation, but remains limited by transgene loss and genotoxicity. Our study aims to test an AAV vector with a promoterless integrating cassette, designed to provide sustained hepatic transgene expression and reduced toxicity in comparison to canonical AAV therapy. APPROACH AND RESULTS: Our AAV vector was designed to insert a methylmalonyl-CoA mutase (MMUT) transgene into the 3' end of the albumin locus and tested in mouse models of methylmalonic acidemia (MMA). After neonatal delivery, we longitudinally evaluated hepatic transgene expression, plasma levels of methylmalonate, and the MMA biomarker, fibroblast growth factor 21 (Fgf21), as well as integration of MMUT in the albumin locus. At necropsy, we surveyed for AAV-related hepatocellular carcinoma (HCC) in all treated MMA mice and control littermates. AAV-mediated genome editing of MMUT into the albumin locus resulted in permanent hepatic correction in MMA mouse models, which was accompanied by decreased levels of methylmalonate and Fgf21, and improved survival without HCC. With time, levels of transgene expression increased and methylmalonate progressively decreased, whereas the number of albumin-MMUT integrations and corrected hepatocytes in MMA mice increased, but not in similarly treated wild-type animals. Additionally, expression of MMUT in the setting of MMA conferred a selective growth advantage upon edited cells, which potentiates the therapeutic response. CONCLUSIONS: In conclusion, our findings demonstrate that AAV-mediated, promoterless, nuclease-free genome editing at the albumin locus provides safe and durable therapeutic benefit in neonatally treated MMA mice.

Evaluating the Genomic Parameters Governing rAAV-Mediated Homologous Recombination.

Recombinant adeno-associated virus (rAAV) vectors have the unique ability to promote targeted integration of transgenes via homologous recombination at specified genomic sites, reaching frequencies of 0.1%-1%. We studied genomic parameters that influence targeting efficiencies on a large scale. To do this, we generated more than 1,000 engineered, doxycycline-inducible target sites in the human HAP1 cell line and infected this polyclonal population with a library of AAV-DJ targeting vectors, with each carrying a unique barcode. The heterogeneity of barcode integration at each target site provided an assessment of targeting efficiency at that locus. We compared targeting efficiency with and without target site transcription for identical chromosomal positions. Targeting efficiency was enhanced by target site transcription, while chromatin accessibility was associated with an increased likelihood of targeting. ChromHMM chromatin states characterizing transcription and enhancers in wild-type K562 cells were also associated with increased AAV-HR efficiency with and without target site transcription, respectively. Furthermore, the amenability of a site to targeting was influenced by the endogenous transcriptional level of intersecting genes. These results define important parameters that may not only assist in designing optimal targeting vectors for genome editing, but also provide new insights into the mechanism of AAV-mediated homologous recombination.

Improved Genome Editing through Inhibition of FANCM and Members of the BTR Dissolvase Complex.

Recombinant adeno-associated virus (rAAV) vectors have the unique property of being able to perform genomic targeted integration (TI) without inducing a double-strand break (DSB). In order to improve our understanding of the mechanism behind TI mediated by AAV and improve its efficiency, we performed an unbiased genetic screen in human cells using a promoterless AAV-homologous recombination (AAV-HR) vector system. We identified that the inhibition of the Fanconi anemia complementation group M (FANCM) protein enhanced AAV-HR-mediated TI efficiencies in different cultured human cells by ∼6- to 9-fold. The combined knockdown of the FANCM and two proteins also associated with the FANCM complex, RecQ-mediated genome instability 1 (RMI1) and Bloom DNA helicase (BLM) from the BLM-topoisomerase IIIα (TOP3A)-RMI (BTR) dissolvase complex (RMI1, having also been identified in our screen), led to the enhancement of AAV-HR-mediated TI up to ∼17 times. AAV-HR-mediated TI in the presence of a nuclease (CRISPR-Cas9) was also increased by ∼1.5- to 2-fold in FANCM and RMI1 knockout cells, respectively. Furthermore, knockdown of FANCM in human CD34+ hematopoietic stem and progenitor cells (HSPCs) increased AAV-HR-mediated TI by ∼3.5-fold. This study expands our knowledge on the mechanisms related to AAV-mediated TI, and it highlights new pathways that might be manipulated for future improvements in AAV-HR-mediated TI.

An orange calcium-modulated bioluminescent indicator for non-invasive activity imaging.

Fluorescent indicators are used widely to visualize calcium dynamics downstream of membrane depolarization or G-protein-coupled receptor activation, but are poorly suited for non-invasive imaging in mammals. Here, we report a bright calcium-modulated bioluminescent indicator named Orange CaMBI (Orange Calcium-modulated Bioluminescent Indicator). Orange CaMBI reports calcium dynamics in single cells and, in the context of a transgenic mouse, reveals calcium oscillations in whole organs in an entirely non-invasive manner.

Transfer RNA-Derived Small RNAs: Another Layer of Gene Regulation and Novel Targets for Disease Therapeutics.

Decades after identification as essential for protein synthesis, transfer RNAs (tRNAs) have been implicated in various cellular processes beyond translation. tRNA-derived small RNAs (tsRNAs), referred to as tRNA-derived fragments (tRFs) or tRNA-derived, stress-induced RNAs (tiRNAs), are produced by cleavage at different sites from mature or pre-tRNAs. They are classified into six major types representing potentially thousands of unique sequences and have been implicated to play a wide variety of regulatory roles in maintaining normal homeostasis, cancer cell viability, tumorigenesis, ribosome biogenesis, chromatin remodeling, translational regulation, intergenerational inheritance, retrotransposon regulation, and viral replication. However, the detailed mechanisms governing these processes remain unknown. Aberrant expression of tsRNAs is found in various human disease conditions, suggesting that a further understanding of the regulatory role of tsRNAs will assist in identifying novel biomarkers, potential therapeutic targets, and gene-regulatory tools. Here, we highlight the classification, biogenesis, and biological role of tsRNAs in regulatory mechanisms of normal and disease states.

Outcome of Femoral-popliteal Bypass Procedures in Different Ethnic Groups in England: A Retrospective Analysis of Hospital Episode Statistics.

BACKGROUND: Previous studies, mainly from the United States, have reported worse outcomes from lower limb bypass procedures in ethnic minority populations. Limited nationwide data are available from ethnic minority populations from Europe. The aim of this study is to investigate outcomes from lower limb bypass procedures in ethnic minorities from England. METHODS: We enquired the "Hospital Episode Statistics" database, using ICD-10 codes to identify all cases of femoral-popliteal bypass operations from English NHS Hospitals from 01/01/2006 to 31/12/2015. Every case was followed up for 2 years for subsequent events. The primary outcomes were mortality and major leg amputation. Patients were broadly categorised according to Black, Asian and White ethnicity. Chi-square test was used to the ethnic groups and odds ratios (OR) were calculated using White ethnic group with the largest numbers of participants as a reference category. RESULTS: In the examined 10-year period, 20825 femoral-popliteal bypass procedures (250 of Black, 167 of Asian, and 20.408 of White ethnicity) were recorded. Thirty-day and 2-year mortality were 2.8% and 16.8% with no significant ethnic differences. Patients of Black ethnicity had higher risk of limb loss compared to Whites (23.2% vs. 15.6%, OR = 1.63, 95% confidence interval (CI) 1.21-2.19, P < 0.01). There was no significant difference in amputation rates between Asians and Whites (16.2% vs.. 15.6%, P = 0.94). CONCLUSIONS: Patients of Black ethnicity are at higher risk of limb loss after a femoropopliteal bypass procedure. Further research is needed to identify the causes of this discrepancy.

Allele-Specific Silencing Ameliorates Restrictive Cardiomyopathy Attributable to a Human Myosin Regulatory Light Chain Mutation.

BACKGROUND: Restrictive cardiomyopathy is a rare heart disease associated with mutations in sarcomeric genes and with phenotypic overlap with hypertrophic cardiomyopathy. There is no approved therapy directed at the underlying cause. Here, we explore the potential of an interfering RNA (RNAi) therapeutic for a human sarcomeric mutation in MYL2 causative of restrictive cardiomyopathy in a mouse model. METHODS: A short hairpin RNA (M7.8L) was selected from a pool for specificity and efficacy. Two groups of myosin regulatory light chain N47K transgenic mice were injected with M7.8L packaged in adeno-associated virus 9 at 3 days of age and 60 days of age. Mice were subjected to treadmill exercise and echocardiography after treatment to determine maximal oxygen uptake and left ventricular mass. At the end of treatment, heart, lung, liver, and kidney tissue was harvested to determine viral tropism and for transcriptomic and proteomic analysis. Cardiomyocytes were isolated for single-cell studies. RESULTS: A one-time injection of AAV9-M7.8L RNAi in 3-day-old humanized regulatory light chain mutant transgenic mice silenced the mutated allele (RLC-47K) with minimal effects on the normal allele (RLC-47N) assayed at 16 weeks postinjection. AAV9-M7.8L RNAi suppressed the expression of hypertrophic biomarkers, reduced heart weight, and attenuated a pathological increase in left ventricular mass. Single adult cardiac myocytes from mice treated with AAV9-M7.8L showed partial restoration of contraction, relaxation, and calcium kinetics. In addition, cardiac stress protein biomarkers, such as calmodulin-dependent protein kinase II and the transcription activator Brg1 were reduced, suggesting recovery toward a healthy myocardium. Transcriptome analyses further revealed no significant changes of argonaute (AGO1, AGO2) and endoribonuclease dicer (DICER1) transcripts, and endogenous microRNAs were preserved, suggesting that the RNAi pathway was not saturated. CONCLUSIONS: Our results show the feasibility, efficacy, and safety of RNAi therapeutics directed towards human restrictive cardiomyopathy. This is a promising step toward targeted therapy for a prevalent human disease.

Selection and evaluation of clinically relevant AAV variants in a xenograft liver model.

Recombinant adeno-associated viral (rAAV) vectors have shown early promise in clinical trials. The therapeutic transgene cassette can be packaged in different AAV capsid pseudotypes, each having a unique transduction profile. At present, rAAV capsid serotype selection for a specific clinical trial is based on effectiveness in animal models. However, preclinical animal studies are not always predictive of human outcome. Here, in an attempt to further our understanding of these discrepancies, we used a chimaeric human-murine liver model to compare directly the relative efficiency of rAAV transduction in human versus mouse hepatocytes in vivo. As predicted from preclinical and clinical studies, rAAV2 vectors functionally transduced mouse and human hepatocytes at equivalent but relatively low levels. However, rAAV8 vectors, which are very effective in many animal models, transduced human hepatocytes rather poorly-approximately 20 times less efficiently than mouse hepatocytes. In light of the limitations of the rAAV vectors currently used in clinical studies, we used the same murine chimaeric liver model to perform serial selection using a human-specific replication-competent viral library composed of DNA-shuffled AAV capsids. One chimaeric capsid composed of five different parental AAV capsids was found to transduce human primary hepatocytes at high efficiency in vitro and in vivo, and provided species-selected transduction in primary liver, cultured cells and a hepatocellular carcinoma xenograft model. This vector is an ideal clinical candidate and a reagent for gene modification of human xenotransplants in mouse models of human diseases. More importantly, our results suggest that humanized murine models may represent a more precise approach for both selecting and evaluating clinically relevant rAAV serotypes for gene therapeutic applications.

Bioengineered AAV Capsids with Combined High Human Liver Transduction In Vivo and Unique Humoral Seroreactivity.

Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications.

Regulated complex assembly safeguards the fidelity of Sleeping Beauty transposition.

The functional relevance of the inverted repeat structure (IR/DR) in a subgroup of the Tc1/mariner superfamily of transposons has been enigmatic. In contrast to mariner transposition, where a topological filter suppresses single-ended reactions, the IR/DR orchestrates a regulatory mechanism to enforce synapsis of the transposon ends before cleavage by the transposase occurs. This ordered assembly process shepherds primary transposase binding to the inner 12DRs (where cleavage does not occur), followed by capture of the 12DR of the other transposon end. This extra layer of regulation suppresses aberrant, potentially genotoxic recombination activities, and the mobilization of internally deleted copies in the IR/DR subgroup, including Sleeping Beauty (SB). In contrast, internally deleted sequences (MITEs) are preferred substrates of mariner transposition, and this process is associated with the emergence of Hsmar1-derived miRNA genes in the human genome. Translating IR/DR regulation to in vitro evolution yielded an SB transposon version with optimized substrate recognition (pT4). The ends of SB transposons excised by a K248A excision+/integration- transposase variant are processed by hairpin resolution, representing a link between phylogenetically, and mechanistically different recombination reactions, such as V(D)J recombination and transposition. Such variants generated by random mutation might stabilize transposon-host interactions or prepare the transposon for a horizontal transfer.