Severe hemophilia A (HA) is an inherited bleeding disorder characterized by <1% of residual factor VIII (FVIII) clotting activity. The disease affects several mammals including dogs, and, like humans, is associated with high morbidity and mortality. In gene therapy using adeno-associated viral (AAV) vectors, the canine model has been one of the best predictors of the therapeutic dose tested in clinical trials for hemophilia B (factor IX deficiency) and other genetic diseases, such as congenital blindness. Here we report our experience with liver gene therapy with AAV-FVIII in two outbred, privately owned dogs with severe HA that resulted in sustained expression of 1-2% of normal FVIII levels and prevented 90% of expected bleeding episodes. A Thr62Met mutation in the F8 gene was identified in one dog. These data recapitulate the improvement of the disease phenotype in research animals, and in humans, with AAV liver gene therapy for hemophilia B. Our experience is a novel example of the benefits of a relevant preclinical canine model to facilitate both translational studies in humans and improved welfare of privately owned dogs.

Mucopolysaccharidosis VII (MPS VII) is due to deficient β-glucuronidase (GUSB) activity, which leads to accumulation of chondroitin, heparan, and dermatan sulfate glycosaminoglycans in various tissues including those of the spine. Associated spine disease can be due to abnormalities in the vertebrae, the intervertebral disks, or other spine tissues. The goal of this study was to determine if neonatal gene therapy could prevent lumbar spine disease in MPS VII dogs. MPS VII dogs were injected intravenously with a retroviral vector (RV) expressing canine GUSB at 2 to 3 days after birth, which resulted in transduction of hepatocytes that secreted GUSB into blood. Expression was stable for up to 11 years, and mean survival was increased from 0.4 years in untreated dogs to 6.1 years in treated dogs. Despite a profound positive clinical effect, 6-month-old RV-treated MPS VII dogs still had hypoplastic ventral epiphyses with reduced calcification in the lumbar spine, which resulted in a reduced stiffness and increased range of motion that were not improved relative to untreated MPS VII dogs. At six to 11 years of age, ventral vertebrae remained hypoplastic in RV-treated MPS VII dogs, and there was desiccation of the nucleus pulposus in some disks. Histochemical staining demonstrated that disks did not have detectable GUSB activity despite high serum GUSB activity, which is likely due to poor diffusion into this relatively avascular structure. Thus, neonatal gene therapy cannot prevent lumbar spine disease in MPS VII dogs, which predicts that enzyme replacement therapy (ERT) will similarly be relatively ineffective even if started at birth.

Polycomb group proteins mediate heritable transcriptional silencing and function through multiprotein complexes that methylate and ubiquitinate histones. The 600-kDa E(Z)/ESC complex, also known as Polycomb repressive complex 2 (PRC2), specifically methylates histone H3 lysine 27 (H3 K27) through the intrinsic histone methyltransferase (HMTase) activity of the E(Z) SET domain. By itself, E(Z) exhibits no detectable HMTase activity and requires ESC for methylation of H3 K27. The molecular basis for this requirement is unknown. ESC binds directly, via its C-terminal WD repeats (beta-propeller domain), to E(Z). Here, we show that the N-terminal region of ESC that precedes its beta-propeller domain interacts directly with histone H3, thereby physically linking E(Z) to its substrate. We show that when expressed in stable S2 cell lines, an N-terminally truncated ESC (FLAG-ESC61-425), like full-length ESC, is incorporated into complexes with E(Z) and binds to a Ubx Polycomb response element in a chromatin immunoprecipitation assay. However, incorporation of this N-terminally truncated ESC into E(Z) complexes prevents trimethylation of histone H3 by E(Z). We also show that a closely related Drosophila melanogaster paralog of ESC, ESC-like (ESCL), and the mammalian homolog of ESC, EED, also interact with histone H3 via their N termini, indicating that the interaction of ESC with histone H3 is evolutionarily conserved, reflecting its functional importance. Our data suggest that one of the roles of ESC (and ESCL and EED) in PRC2 complexes is to enable E(Z) to utilize histone H3 as a substrate by physically linking enzyme and substrate.

Acid ceramidase is required to maintain the metabolic balance of several important bioactive lipids, including ceramide, sphingosine and sphingosine-1-phosphate. Here we show that addition of recombinant acid ceramidase (rAC) to primary chondrocyte culture media maintained low levels of ceramide and led to elevated sphingosine by 48 hours. Surprisingly, after three weeks of expansion the chondrogenic phenotype of these cells also was markedly improved, as assessed by a combination of histochemical staining (Alcian Blue and Safranin-O), western blotting (e.g., Sox9, aggrecan, collagen 2A1), and/or qPCR. The same effects were evident in rat, equine and human cells, and were observed in monolayer and 3-D cultures. rAC also reduced the number of apoptotic cells in some culture conditions, contributing to overall improved cell quality. In addition to these effects on primary chondrocytes, when rAC was added to freshly harvested rat, equine or feline bone marrow cultures an ~2-fold enrichment of mesenchymal stem cells (MSCs) was observed by one week. rAC also improved the chondrogenic differentiation of MSCs, as revealed by histochemical and immunostaining. These latter effects were synergistic with TGF-beta1. Based on these results we propose that rAC could be used to improve the outcome of cell-based cartilage repair by maintaining the quality of the expanded cells, and also might be useful in vivo to induce endogenous cartilage repair in combination with other techniques. The results also suggest that short-term changes in sphingolipid metabolism may lead to longer-term effects on the chondrogenic phenotype.

Hematopoietic stem cells (HSC) are important targets for gene therapy. Most protocols involve ex vivo modification, in which HSC are transduced in vitro and injected into the recipient. An in vivo delivery method might simplify HSC gene therapy. We previously demonstrated that iv injection of an amphotropic retroviral vector (RV) into newborn mice resulted in long-term expression from hepatocytes. The goal of this study was to determine if HSC were also transduced. After neonatal administration of 1 x 10(10) transducing units/kg of RV, peripheral blood cells had approximately 0.1 copy of RV per cell for up to 22 months. At 18 months, RV sequences were detected in T, B, and myeloid cells from bone marrow (BM). Unfractionated BM was transplanted into naive recipients after total body irradiation. Recipients maintained similar levels of the RV in their blood cells for 10 months, at which time RV sequences were present at the same integration site in all lineages of cells from BM. We conclude that neonatal iv injection of RV results in transduction of HSC in mice, which might be used for BM-directed gene therapy. Transduction of blood cells after liver-directed neonatal gene therapy might have adverse effects in patients, although no leukemias developed here.

The mechanisms that cells use to monitor telomere integrity, and the array of responses that may be induced, are not fully defined. To date there have been no studies in animals describing the ability of cells to survive and contribute to adult organs following telomere loss. We developed assays to monitor the ability of somatic cells to proliferate and differentiate after telomere loss. Here we show that p53 and Chk2 limit the growth and differentiation of cells that lose a telomere. Furthermore, our results show that two copies of the genes encoding p53 and Chk2 are required for the cell to mount a rapid wildtype response to a missing telomere. Finally, our results show that, while Chk2 functions by activating the p53-dependent apoptotic cascade, Chk2 also functions independently of p53 to limit survival. In spite of these mechanisms to eliminate cells that have lost a telomere, we find that such cells can make a substantial contribution to differentiated adult tissues.

Corneal transparency is maintained, in part, by specialized fibroblasts called keratocytes, which reside in the fibrous lamellae of the stroma. Corneal clouding, a condition that impairs visual acuity, is associated with numerous diseases, including mucopolysaccharidosis (MPS) type VII. MPS VII is due to deficiency in β-glucuronidase (β-glu) enzymatic activity, which leads to accumulation of glycosaminoglycans (GAGs), and secondary accumulation of gangliosides. Here, we tested the efficacy of canine adenovirus type 2 (CAV-2) vectors to transduce keratocyte in vivo in mice and nonhuman primates, and ex vivo in dog and human corneal explants. Following efficacy studies, we asked if we could treat corneal clouding by the injection a helper-dependent (HD) CAV-2 vector (HD-RIGIE) harboring the human cDNA coding for β-glu (GUSB) in the canine MPS VII cornea. β-Glu activity, GAG content, and lysosome morphology and physiopathology were analyzed. We found that HD-RIGIE injections efficiently transduced coxsackievirus adenovirus receptor-expressing keratocytes in the four species and, compared to mock-injected controls, improved the pathology in the canine MPS VII cornea. The key criterion to corrective therapy was the steady controlled release of β-glu and its diffusion throughout the collagen-dense stroma. These data support the continued evaluation of HD CAV-2 vectors to treat diseases affecting corneal keratocytes.

Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease arising from mutations in β-d-glucuronidase (GUSB), which results in glycosaminoglycan (GAG) accumulation and a variety of clinical manifestations including neurological disease. Herein, MPS VII dogs were injected intravenously (i.v.) and/or intrathecally (i.t.) via the cisterna magna with AAV9 or AAVrh10 vectors carrying the canine GUSB cDNA. Although i.v. injection alone at 3 days of age resulted in normal cerebrospinal fluid (CSF) GUSB activity, brain tissue homogenates had only ~1 to 6% normal GUSB activity and continued to have elevated GAG storage. In contrast, i.t. injection at 3 weeks of age resulted in CSF GUSB activity 44-fold normal while brain tissue homogenates had >100% normal GUSB activity and reduced GAGs compared with untreated dogs. Markers for secondary storage and inflammation were eliminated in i.t.-treated dogs and reduced in i.v.-treated dogs compared with untreated dogs. Given that i.t.-treated dogs expressed higher levels of GUSB in the CNS tissues compared to those treated i.v., we conclude that i.t. injection of AAV9 or AAVrh10 vectors is more effective than i.v. injection alone in the large animal model of MPS VII.

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient activity of β-glucuronidase, leading to progressive accumulation of incompletely degraded heparan, dermatan, and chondroitin sulfate glycosaminoglycans (GAGs). Patients with MPS VII exhibit progressive skeletal deformity including kyphoscoliosis and joint dysplasia, which decrease quality of life and increase mortality. Previously, using the naturally-occurring canine model, we demonstrated that one of the earliest skeletal abnormalities to manifest in MPS VII is failed initiation of secondary ossification in vertebrae and long bones at the requisite postnatal developmental stage. The objective of this study was to obtain global insights into the molecular mechanisms underlying this failed initiation of secondary ossification. Epiphyseal tissue was isolated postmortem from the vertebrae of control and MPS VII-affected dogs at 9 and 14 days-of-age (n = 5 for each group). Differences in global gene expression across this developmental window for both cohorts were measured using whole-transcriptome sequencing (RNA-Seq). Principal Component Analysis revealed clustering of samples within each group, indicating clear effects of both age and disease state. At 9 days-of-age, 1375 genes were significantly differentially expressed between MPS VII and control, and by 14 days-of-age, this increased to 4719 genes. A targeted analysis focused on signaling pathways important in the regulation of endochondral ossification was performed, and a subset of gene expression differences were validated using qPCR. Osteoactivin (GPNMB) was the top upregulated gene in MPS VII at both ages. In control samples, temporal changes in gene expression from 9 to 14 days-of-age were consistent with chondrocyte maturation, cartilage resorption, and osteogenesis. In MPS VII samples, however, elements of key osteogenic pathways such as Wnt/β-catenin and BMP signaling were not upregulated during this same developmental window suggesting that important bone formation pathways are not activated. In conclusion, this study represents an important step towards identifying therapeutic targets and biomarkers for bone disease in MPS VII patients during postnatal growth.

Adeno-associated viral (AAV) vectors have emerged as the preferred platform for in vivo gene transfer because of their combined efficacy and safety. However, insertional mutagenesis with the subsequent development of hepatocellular carcinomas (HCCs) has been recurrently noted in newborn mice treated with high doses of AAV, and more recently, the association of wild-type AAV integrations in a subset of human HCCs has been documented. Here, we address, in a comprehensive, prospective study, the long-term risk of tumorigenicity in young adult mice following delivery of single-stranded AAVs targeting liver. HCC incidence in mice treated with therapeutic and reporter AAVs was low, in contrast to what has been previously documented in mice treated as newborns with higher doses of AAV. Specifically, HCCs developed in 6 out 76 of AAV-treated mice, and a pathogenic integration of AAV was found in only one tumor. Also, no evidence of liver tumorigenesis was found in juvenile AAV-treated mucopolysaccharidosis type VI (MPS VI) cats followed as long as 8 years after vector administration. Together, our results support the low risk of tumorigenesis associated with AAV-mediated gene transfer targeting juvenile/young adult livers, although constant monitoring of subjects enrolled in AAV clinical trial is advisable.

Mucopolysaccharidosis type VII (MPS VII) is characterized by deficient β-glucuronidase (GUSB) activity, which leads to accumulation of chondroitin, heparan and dermatan sulfate glycosaminoglycans (GAGs), and multisystemic disease. MPS VII patients can develop kypho-scoliotic deformity and spinal cord compression due to disease of intervertebral disks, vertebral bodies, and associated tissues. We have previously demonstrated in MPS VII dogs that intervertebral disks degenerate, vertebral bodies have irregular surfaces, and vertebral body epiphyses have reduced calcification, but the pathophysiological mechanisms underlying these changes are unclear. We hypothesized that some of these manifestations could be due to upregulation of destructive proteases, possibly via the binding of GAGs to Toll-like receptor 4 (TLR4), as has been proposed for other tissues in MPS models. In this study, the annulus fibrosus of the intervertebral disk of 6-month-old MPS VII dogs had cathepsin B and K activities that were 117- and 2-fold normal, respectively, which were associated with elevations in mRNA levels for these cathepsins as well as TLR4. The epiphyses of MPS VII dogs had a marked elevation in mRNA for the cartilage-associated gene collagen II, consistent with a developmental delay in the conversion of the cartilage to bone in this region. The spine obtained at autopsy from a young man with MPS VII exhibited similar increased cartilage in the vertebral bodies adjacent to the end plates, disorganization of the intervertebral disks, and irregular vertebral end plate morphology. These data suggest that the pathogenesis of destructive changes in the spine in MPS VII may involve upregulation of cathepsins. Inhibition of destructive proteases, such as cathepsins, might reduce spine disease in patients with MPS VII or related disorders.

Deficiency of glycogen branching enzyme (GBE) activity causes glycogen storage disease type IV (GSD IV), an autosomal recessive error of metabolism. Abnormal glycogen accumulates in myocytes, hepatocytes, and neurons, causing variably progressive, benign to lethal organ dysfunctions. A naturally occurring orthologue of human GSD IV was described previously in Norwegian forest cats (NFC). Here, we report that while most affected kittens die at or soon after birth, presumably due to hypoglycemia, survivors of the perinatal period appear clinically normal until onset of progressive neuromuscular degeneration at 5 months of age. Molecular investigation of affected cats revealed abnormally spliced GBE1 mRNA products and lack of GBE cross-reactive material in liver and muscle. Affected cats are homozygous for a complex rearrangement of genomic DNA in GBE1, constituted by a 334 bp insertion at the site of a 6.2 kb deletion that extends from intron 11 to intron 12 (g. IVS11+1552_IVS12-1339 del6.2kb ins334 bp), removing exon 12. An allele-specific, PCR-based test demonstrates that the rearrangement segregates with the disease in the GSD IV kindred and is not found in unrelated normal cats. Screening of 402 privately owned NFC revealed 58 carriers and 4 affected cats. The molecular characterization of feline GSD IV will enhance further studies of GSD IV pathophysiology and development of novel therapies in this unique animal model.

Mucopolysaccharidosis I (MPS I) and MPS VII are due to deficient activity of the glycosaminoglycan-degrading lysosomal enzymes alpha-L-iduronidase and beta-glucuronidase, respectively, and result in abnormal bones and joints. Here, the severity of skeletal disease in MPS I and MPS VII dogs and the effects of neonatal gene therapy were evaluated. For untreated MPS VII dogs, the lengths of the second cervical vertebrae (C2) and the femur were only 56% and 84% of normal, respectively, and bone dysplasia and articular erosions, and joint subluxation were severe. Previously, we reported that neonatal intravenous injection of a retroviral vector (RV) with the appropriate gene resulted in expression in liver and blood cells, and high serum enzyme activity. In this study, we demonstrate that C2 and femurs of RV-treated MPS VII dogs were longer at 82% and 101% of normal, respectively, and there were partial improvements of qualitative abnormalities. For untreated MPS I dogs, the lengths of C2 and femurs (91% and 96% of normal, respectively) were not significantly different from normal dogs. Qualitative changes in MPS I bones and joints were generally modest and were partially improved with RV treatment, although cervical spine disease was severe and was difficult to correct with gene therapy in both models. The greater severity of skeletal disease in MPS VII than in MPS I dogs may reflect accumulation of chondroitin sulfate in cartilage in MPS VII, or could relate to the specific mutations. Neonatal RV-mediated gene therapy ameliorates, but does not prevent, skeletal disease in MPS I and MPS VII dogs.

The mucopolysaccharidoses (MPS) are a family of lysosomal storage diseases resulting in developmental defects and, in some types, mental retardation and other neurological symptoms. To gain insight into the neurological dysfunction in MPS, we examined the morphology of olfactory epithelia (OE) and physiology of olfactory receptor neurons (ORNs) in cat models of MPS I, a type in which neuronal lesions are prominent, and MPS VI, in which they are essentially absent. Histopathology showed that both groups of MPS-affected cats had significantly thinner OE than controls. Although immature and mature ORNs were present in both MPS I and VI affected OE, the OE of MPS I-affected cats was structurally disorganized. ORN function was assessed with calcium imaging and patch-clamp recording. Few viable ORNs were recovered from MPS VI cats, but these exhibited normal responses to odors and pharmacological stimuli. In contrast, viable ORNs were as prevalent in MPS I as in controls but were significantly less likely to respond to odor stimuli, although other responses were normal. Disrupted OE organization and impaired ORN function in MPS I, but not MPS VI, corresponds to the central nervous system lesions found in MPS I but not MPS VI. These data represent the first neurophysiological correlate of this correspondence and have implications both for understanding the role of glycosaminoglycans in maintenance of the OE and for targeting further research into the basis for and treatment of the neurological consequences of MPS disorders.

Mucopolysaccharidosis VII (MPS VII) is due to the deficient activity of β-glucuronidase (GUSB) and results in the accumulation of glycosaminoglycans (GAGs) in lysosomes and multisystemic disease with cardiovascular manifestations. The goal here was to determine the pathogenesis of mitral valve (MV) disease in MPS VII dogs. Untreated MPS VII dogs had a marked reduction in the histochemical signal for structurally-intact collagen in the MV at 6 months of age, when mitral regurgitation had developed. Electron microscopy demonstrated that collagen fibrils were of normal diameter, but failed to align into large parallel arrays. mRNA analysis demonstrated a modest reduction in the expression of genes that encode collagen or collagen-associated proteins such as the proteoglycan decorin which helps collagen fibrils assemble, and a marked increase for genes that encode proteases such as cathepsins. Indeed, enzyme activity for cathepsin B (CtsB) was 19-fold normal. MPS VII dogs that received neonatal intravenous injection of a gamma retroviral vector had an improved signal for structurally-intact collagen, and reduced CtsB activity relative to that seen in untreated MPS VII dogs. We conclude that MR in untreated MPS VII dogs was likely due to abnormalities in MV collagen structure. This could be due to upregulation of enzymes that degrade collagen or collagen-associated proteins, to the accumulation of GAGs that compete with proteoglycans such as decorin for binding to collagen, or to other causes. Further delineation of the etiology of abnormal collagen structure may lead to treatments that improve biomechanical properties of the MV and other tissues.

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disease due to deficient activity of β-glucuronidase (GUSB), and results in glycosaminoglycan accumulation. Skeletal manifestations include bone dysplasia, degenerative joint disease, and growth retardation. One gene therapy approach for MPS VII involves neonatal intravenous injection of a gamma retroviral vector expressing GUSB, which results in stable expression in liver and secretion of enzyme into blood at levels predicted to be similar or higher to enzyme replacement therapy. The goal of this study was to evaluate the long-term effect of neonatal gene therapy on skeletal manifestations in MPS VII dogs. Treated MPS VII dogs could walk throughout their lives, while untreated MPS VII dogs could not stand beyond 6 months and were dead by 2 years. Luxation of the coxofemoral joint and the patella, dysplasia of the acetabulum and supracondylar ridge, deep erosions of the distal femur, and synovial hyperplasia were reduced, and the quality of articular bone was improved in treated dogs at 6 to 11 years of age compared with untreated MPS VII dogs at 2 years or less. However, treated dogs continued to have osteophyte formation, cartilage abnormalities, and an abnormal gait. Enzyme activity was found near synovial blood vessels, and there was 2% as much GUSB activity in synovial fluid as in serum. We conclude that neonatal gene therapy reduces skeletal abnormalities in MPS VII dogs, but clinically-relevant abnormalities remain. Enzyme replacement therapy will probably have similar limitations long-term.

Mucopolysaccharidosis (MPS) VI is due to a deficiency in the activity of N-acetylgalactosamine 4-sulfatase (4S), also known as arylsulfatase B. Previously, retroviral vector (RV)-mediated neonatal gene therapy reduced the clinical manifestations of MPS I and MPS VII in mice and dogs. However, sulfatases require post-translational modification by sulfatase-modifying factors. MPS VI cats were injected intravenously (i.v.) with a gamma RV-expressing feline 4S, resulting in 5 ± 3 copies of RV per 100 cells in liver. Liver and serum 4S activity were 1,450 ± 1,720 U/mg (26-fold normal) and 107 ± 60 U/ml (13-fold normal), respectively, and were directly proportional to the liver 4S protein levels for individual cats. This study suggests that sulfatase-modifying factor (SUMF) activity in liver was sufficient to result in active enzyme despite overexpression of 4S. RV-treated MPS VI cats achieved higher body weights and longer appendicular skeleton lengths, had reduced articular cartilage erosion, and reduced aortic valve thickening and aortic dilatation compared with untreated MPS VI cats, although cervical vertebral bone lengths were not improved. This demonstrates that therapeutic expression of a functional sulfatase protein can be achieved with neonatal gene therapy using a gamma RV, but some aspects of bone disease remain difficult to treat.

The Drosophila esc-like gene (escl) encodes a protein very similar to ESC. Like ESC, ESCL binds directly to the E(Z) histone methyltransferase via its WD region. In contrast to ESC, which is present at highest levels during embryogenesis and low levels thereafter, ESCL is continuously present throughout development and in adults. ESC/E(Z) complexes are present at high levels mainly during embryogenesis but ESCL/E(Z) complexes are found throughout development. While depletion of either ESCL or ESC by RNAi in S2 and Kc cells has little effect on E(Z)-mediated methylation of histone H3 lysine 27 (H3K27), simultaneous depletion of ESCL and ESC results in loss of di- and trimethyl-H3K27, indicating that either ESC or ESCL is necessary and sufficient for di- and trimethylation of H3K27 in vivo. While E(Z) complexes in S2 cells contain predominantly ESC, in ESC-depleted S2 cells, ESCL levels rise dramatically and ESCL replaces ESC in E(Z) complexes. A mutation in escl that produces very little protein is viable and exhibits no phenotypes but strongly enhances esc mutant phenotypes, suggesting they have similar functions. esc escl double homozygotes die at the end of the larval period, indicating that the well-known "maternal rescue" of esc homozygotes requires ESCL. Furthermore, maternal and zygotic over-expression of escl fully rescues the lethality of esc null mutant embryos that contain no ESC protein, indicating that ESCL can substitute fully for ESC in vivo. These data thus indicate that ESC and ESCL play similar if not identical functions in E(Z) complexes in vivo. Despite this, when esc is expressed normally, escl appears to be entirely dispensable, at least for development into morphologically normal fertile adults. Furthermore, the larval lethality of esc escl double mutants, together with the lack of phenotypes in the escl mutant, further suggests that in wild-type (esc(+)) animals it is the post-embryonic expression of esc, not escl, that is important for development of normal adults. Thus escl appears to function in a backup capacity during development that becomes important only when normal esc expression is compromised.

The addition of a new telomere onto a chromosome break, a process termed healing, has been studied extensively in organisms that utilize telomerase to maintain their telomeres. In comparison, relatively little is known about how new telomeres are constructed on broken chromosomes in organisms that do not use telomerase. Chromosome healing was studied in somatic and germline cells of Drosophila melanogaster, a nontelomerase species. We observed, for the first time, that broken chromosomes can be healed in somatic cells. In addition, overexpression of the telomere cap component Hiphop increased the survival of somatic cells with broken chromosomes, while the cap component HP1 did not, and overexpression of the cap protein HOAP decreased their survival. In the male germline, Hiphop overexpression greatly increased the transmission of healed chromosomes. These results indicate that Hiphop can stimulate healing of a chromosome break. We suggest that this reflects a unique function of Hiphop: it is capable of seeding formation of a new telomeric cap on a chromosome end that lacks a telomere.