All Publications

  • AAV-mediated genome editing is influenced by the formation of R-loops. bioRxiv : the preprint server for biology Puzzo, F., Crossley, M. P., Goswami, A., Zhang, F., Pekrun, K., Garzon, J. L., Cimprich, K. A., Kay, M. A. 2024


    Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone, 3' end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop- deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.

    View details for DOI 10.1101/2024.05.07.592855

    View details for PubMedID 38766176

  • Generation of DNA Aptamers Against Pancreatic Surface Receptors Towards AAV Cell-Specific Targeting Trisno, S. L., Puzzo, F., Kay, M. A. CELL PRESS. 2023: 228
  • The Role of DNA/RNA Hybrids on the Efficiency of Nuclease-Free Genome Editing and AAV Integration <i>In Vivo</i> Puzzo, F., Crossley, M. P., Pekrun, K., Goswami, A., Zhang, F., Cimprich, K., Kay, M. A. CELL PRESS. 2023: 518
  • Promoterless AAV Gene Targeting Vectors Favor Integration into the Albumin Locus Regardless of Homology Arm Sequences Pekrun, K., Stephens, C., Zhang, F., Jang, H., Puzzo, F., Liehr, P., Goswami, A., Kay, M. A. CELL PRESS. 2023: 738-739
  • Aptamer-programmable adeno-associated viral vectors as a novel platform for cell-specific gene transfer. Molecular therapy. Nucleic acids Puzzo, F., Zhang, C., Powell Gray, B., Zhang, F., Sullenger, B. A., Kay, M. A. 2023; 31: 383-397


    Adeno-associated viruses (AAVs) are commonly used for invivo gene therapy. Nevertheless, the wide tropism that characterizes these vectors limits specific targeting to a particular cell type or tissue. Here, we developed new chemically modified AAV vectors (Nepsilon-AAVs) displaying a single site substitution on the capsid surface for post-production vector engineering through biorthogonal copper-free click chemistry. We were able to identify AAV vectors that would tolerate the unnatural amino acid substitution on the capsid without disrupting their packaging efficiency. We functionalized the Nepsilon-AAVs through conjugation with DNA (AS1411) or RNA (E3) aptamers or with a folic acid moiety (FA). E3-, AS1411-, and FA-AAVs showed on average a 3- to 9-fold increase in transduction compared with their non-conjugated counterparts in different cancer cell lines. Using specific competitors, we established ligand-specific transduction. Invivo studies confirmed the selective uptake of FA-AAV and AS1411-AAV without off-target transduction in peripheral organs. Overall, the high versatility of these novel Nepsilon-AAVs might pave the way to tailoring gene therapy vectors toward specific types of cells both for exvivo and invivo applications.

    View details for DOI 10.1016/j.omtn.2023.01.007

    View details for PubMedID 36817723

  • Loops as Determinant of AAV Integration by Homologous Recombination Puzzo, F., Zhang, F., Pekrun, K., Kay, M. A. CELL PRESS. 2022: 90-91
  • Programmable Adeno-Associated Viral Vectors for Cell Specific Targeting Puzzo, F., Zhang, C., Zhang, F., Kay, M. A. CELL PRESS. 2022: 66
  • Promoterless AAV Vectors with Homology Arms Can Integrate and Express from Transcriptionally Active Sites in Non-Targeted Loci Stephens, C. J., Pekrun, K., Xu, J., Tsuji, S., Jing, Y., Puzzo, F., Zhang, F., Kay, M. A. CELL PRESS. 2022: 391-392
  • Improved Genome Editing through Inhibition of FANCM and Members of the BTR Dissolvase Complex. Molecular therapy : the journal of the American Society of Gene Therapy de Alencastro, G. n., Puzzo, F. n., Pavel-Dinu, M. n., Zhang, F. n., Pillay, S. n., Majzoub, K. n., Tiffany, M. n., Jang, H. n., Sheikali, A. n., Cromer, M. K., Meetei, R. n., Carette, J. E., Porteus, M. H., Pekrun, K. n., Kay, M. A. 2021; 29 (3): 1016–27


    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.

    View details for DOI 10.1016/j.ymthe.2020.10.020

    View details for PubMedID 33678249

  • Rescue of Advanced Pompe Disease in Mice with Hepatic Expression of Secretable Acid α-Glucosidase. Molecular therapy : the journal of the American Society of Gene Therapy Cagin, U., Puzzo, F., Gomez, M. J., Moya-Nilges, M., Sellier, P., Abad, C., Van Wittenberghe, L., Daniele, N., Guerchet, N., Gjata, B., Collaud, F., Charles, S., Sola, M. S., Boyer, O., Krijnse-Locker, J., Ronzitti, G., Colella, P., Mingozzi, F. 2020; 28 (9): 2056-2072


    Pompe disease is a neuromuscular disorder caused by disease-associated variants in the gene encoding for the lysosomal enzyme acid α-glucosidase (GAA), which converts lysosomal glycogen to glucose. We previously reported full rescue of Pompe disease in symptomatic 4-month-old Gaa knockout (Gaa-/-) mice by adeno-associated virus (AAV) vector-mediated liver gene transfer of an engineered secretable form of GAA (secGAA). Here, we showed that hepatic expression of secGAA rescues the phenotype of 4-month-old Gaa-/- mice at vector doses at which the native form of GAA has little to no therapeutic effect. Based on these results, we then treated severely affected 9-month-old Gaa-/- mice with an AAV vector expressing secGAA and followed the animals for 9 months thereafter. AAV-treated Gaa-/- mice showed complete reversal of the Pompe phenotype, with rescue of glycogen accumulation in most tissues, including the central nervous system, and normalization of muscle strength. Transcriptomic profiling of skeletal muscle showed rescue of most altered pathways, including those involved in mitochondrial defects, a finding supported by structural and biochemical analyses, which also showed restoration of lysosomal function. Together, these results provide insight into the reversibility of advanced Pompe disease in the Gaa-/- mouse model via liver gene transfer of secGAA.

    View details for DOI 10.1016/j.ymthe.2020.05.025

    View details for PubMedID 32526204

    View details for PubMedCentralID PMC7474269

  • Proteins Complex of the Fanconi Anemia Pathway as Determinant of AAV-Mediated Genomic Targeted Integration Puzzo, F., de Alencastro, G., Pavel-Dinu, M., Zhang, F., Pillay, S., Majzoub, K., Tiffany, M., Jang, H., Sheikali, A., Cromer, K. M., Meetei, R., Carette, J. E., Porteus, M. H., Pekrun, K., Kay, M. A. CELL PRESS. 2020: 459
  • Exploiting the Regenerative Capacity of Liver for Nuclease-Free Genome Editing Puzzo, F., De Alencastro, G., Patijn, G., Zhang, F., Pekrun, K., Kay, M. CELL PRESS. 2019: 463
  • AAV Gene Transfer with Tandem Promoter Design Prevents Anti-transgene Immunity and Provides Persistent Efficacy in Neonate Pompe Mice MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT Colella, P., Sellier, P., Verdera, H., Puzzo, F., van Wittenberghe, L., Guerchet, N., Daniele, N., Gjata, B., Marmier, S., Charles, S., Sola, M., Ragone, I., Leborgne, C., Collaud, F., Mingozzi, F. 2019; 12: 85–101


    Hepatocyte-restricted, AAV-mediated gene transfer is being used to provide sustained, tolerogenic transgene expression in gene therapy. However, given the episomal status of the AAV genome, this approach cannot be applied to pediatric disorders when hepatocyte proliferation may result in significant loss of therapeutic efficacy over time. In addition, many multi-systemic diseases require widespread expression of the therapeutic transgene that, when provided with ubiquitous or tissue-specific non-hepatic promoters, often results in anti-transgene immunity. Here we have developed tandem promoter monocistronic expression cassettes that, packaged in a single AAV, provide combined hepatic and extra-hepatic tissue-specific transgene expression and prevent anti-transgene immunity. We validated our approach in infantile Pompe disease, a prototype disease caused by lack of the ubiquitous enzyme acid-alpha-glucosidase (GAA), presenting multi-systemic manifestations and detrimental anti-GAA immunity. We showed that the use of efficient tandem promoters prevents immune responses to GAA following systemic AAV gene transfer in immunocompetent Gaa-/- mice. Then we demonstrated that neonatal gene therapy with either AAV8 or AAV9 in Gaa-/- mice resulted in persistent therapeutic efficacy when using a tandem liver-muscle promoter (LiMP) that provided high and persistent transgene expression in non-dividing extra-hepatic tissues. In conclusion, the tandem promoter design overcomes important limitations of AAV-mediated gene transfer and can be beneficial when treating pediatric conditions requiring persistent multi-systemic transgene expression and prevention of anti-transgene immunity.

    View details for DOI 10.1016/j.omtm.2018.11.002

    View details for Web of Science ID 000461055600007

    View details for PubMedID 30581888

    View details for PubMedCentralID PMC6299151

  • Targeting Mitochondrial Defects to Increase Longevity in Animal Models of Neurodegenerative Diseases REVIEWS ON BIOMARKER STUDIES OF METABOLIC AND METABOLISM-RELATED DISORDERS Casajus Pelegay, E., Puzzo, F., Yilmazer, A., Cagin, U., Guest, P. C. 2019; 1134: 89–110
  • Targeting Mitochondrial Defects to Increase Longevity in Animal Models of Neurodegenerative Diseases. Advances in experimental medicine and biology Casajus Pelegay, E., Puzzo, F., Yilmazer, A., Cagin, U. 2019; 1134: 89–110


    Bioenergetic homeostasis is a vital process maintaining cellular health and has primary importance in neuronal cells due to their high energy demand markedly at synapses. Mitochondria, the metabolic hubs of the cells, are the organelles responsible for producing energy in the form of ATP by using nutrients and oxygen. Defects in mitochondrial homeostasis result in energy deprivation and can lead to disrupted neuronal functions. Mitochondrial defects adversely contribute to the pathogenesis of neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's disease (PD). Mitochondrial defects not only include reduced ATP levels but also increased reactive oxygen species (ROS) leading to cellular damage. Here, we detail the mechanisms that lead to neuronal pathologies involving mitochondrial defects. Furthermore, we discuss how to target these mitochondrial defects in order to have beneficial effects as novel and complementary therapeutic avenues in neurodegenerative diseases. The critical evaluation of these strategies and their potential outcome can pave the way for finding novel therapies for neurodegenerative pathologies.

    View details for PubMedID 30919333

  • Rescue of GSDIII Phenotype with Gene Transfer Requires Liver- and Muscle-Targeted GDE Expression MOLECULAR THERAPY Vidal, P., Pagliarani, S., Colella, P., Verdera, H., Jauze, L., Gjorgjieva, M., Puzzo, F., Marmier, S., Collaud, F., Sola, M., Charles, S., Lucchiari, S., van Wittenberghe, L., Vignaud, A., Gjata, B., Richard, I., Laforet, P., Malfatti, E., Mithieux, G., Rajas, F., Comi, G., Ronzitti, G., Mingozzi, F. 2018; 26 (3): 890–901


    Glycogen storage disease type III (GSDIII) is an autosomal recessive disorder caused by a deficiency of glycogen-debranching enzyme (GDE), which results in profound liver metabolism impairment and muscle weakness. To date, no cure is available for GSDIII and current treatments are mostly based on diet. Here we describe the development of a mouse model of GSDIII, which faithfully recapitulates the main features of the human condition. We used this model to develop and test novel therapies based on adeno-associated virus (AAV) vector-mediated gene transfer. First, we showed that overexpression of the lysosomal enzyme alpha-acid glucosidase (GAA) with an AAV vector led to a decrease in liver glycogen content but failed to reverse the disease phenotype. Using dual overlapping AAV vectors expressing the GDE transgene in muscle, we showed functional rescue with no impact on glucose metabolism. Liver expression of GDE, conversely, had a direct impact on blood glucose levels. These results provide proof of concept of correction of GSDIII with AAV vectors, and they indicate that restoration of the enzyme deficiency in muscle and liver is necessary to address both the metabolic and neuromuscular manifestations of the disease.

    View details for DOI 10.1016/j.ymthe.2017.12.019

    View details for Web of Science ID 000427911800024

    View details for PubMedID 29396266

    View details for PubMedCentralID PMC5910667

  • Rescue of Pompe disease in mice by AAV-mediated liver delivery of secretable acid alpha-glucosidase SCIENCE TRANSLATIONAL MEDICINE Puzzo, F., Colella, P., Biferi, M. G., Bali, D., Paulk, N. K., Vidal, P., Collaud, F., Simon-Sola, M., Charles, S., Hardet, R., Leborgne, C., Meliani, A., Cohen-Tannoudji, M., Astord, S., Gjata, B., Sellier, P., van Wittenberghe, L., Vignaud, A., Boisgerault, F., Barkats, M., Laforet, P., Kay, M. A., Koeberl, D. D., Ronzitti, G., Mingozzi, F. 2017; 9 (418)


    Glycogen storage disease type II or Pompe disease is a severe neuromuscular disorder caused by mutations in the lysosomal enzyme, acid α-glucosidase (GAA), which result in pathological accumulation of glycogen throughout the body. Enzyme replacement therapy is available for Pompe disease; however, it has limited efficacy, has high immunogenicity, and fails to correct pathological glycogen accumulation in nervous tissue and skeletal muscle. Using bioinformatics analysis and protein engineering, we developed transgenes encoding GAA that could be expressed and secreted by hepatocytes. Then, we used adeno-associated virus (AAV) vectors optimized for hepatic expression to deliver the GAA transgenes to Gaa knockout (Gaa-/-) mice, a model of Pompe disease. Therapeutic gene transfer to the liver rescued glycogen accumulation in muscle and the central nervous system, and ameliorated cardiac hypertrophy as well as muscle and respiratory dysfunction in the Gaa-/- mice; mouse survival was also increased. Secretable GAA showed improved therapeutic efficacy and lower immunogenicity compared to nonengineered GAA. Scale-up to nonhuman primates, and modeling of GAA expression in primary human hepatocytes using hepatotropic AAV vectors, demonstrated the therapeutic potential of AAV vector-mediated liver expression of secretable GAA for treating pathological glycogen accumulation in multiple tissues in Pompe disease.

    View details for PubMedID 29187643

    View details for PubMedCentralID PMC5826611

  • Autophagy determines efficiency of liver-directed gene therapy with adeno-associated viral vectors. Hepatology (Baltimore, Md.) Hösel, M., Huber, A., Bohlen, S., Lucifora, J., Ronzitti, G., Puzzo, F., Boisgerault, F., Hacker, U. T., Kwanten, W. J., Klöting, N., Blüher, M., Gluschko, A., Schramm, M., Utermöhlen, O., Bloch, W., Mingozzi, F., Krut, O., Büning, H. 2017; 66 (1): 252-265


    Use of adeno-associated viral (AAV) vectors for liver-directed gene therapy has shown considerable success, particularly in patients with severe hemophilia B. However, the high vector doses required to reach therapeutic levels of transgene expression caused liver inflammation in some patients that selectively destroyed transduced hepatocytes. We hypothesized that such detrimental immune responses can be avoided by enhancing the efficacy of AAV vectors in hepatocytes. Because autophagy is a key liver response to environmental stresses, we characterized the impact of hepatic autophagy on AAV infection. We found that AAV induced mammalian target of rapamycin (mTOR)-dependent autophagy in human hepatocytes. This cell response was critically required for efficient transduction because under conditions of impaired autophagy (pharmacological inhibition, small interfering RNA knockdown of autophagic proteins, or suppression by food intake), recombinant AAV-mediated transgene expression was markedly reduced, both in vitro and in vivo. Taking advantage of this dependence, we employed pharmacological inducers of autophagy to increase the level of autophagy. This resulted in greatly improved transduction efficiency of AAV vectors in human and mouse hepatocytes independent of the transgene, driving promoter, or AAV serotype and was subsequently confirmed in vivo. Specifically, short-term treatment with a single dose of torin 1 significantly increased vector-mediated hepatic expression of erythropoietin in C57BL/6 mice. Similarly, coadministration of rapamycin with AAV vectors resulted in markedly enhanced expression of human acid-α-glucosidase in nonhuman primates.We identified autophagy as a pivotal cell response determining the efficiency of AAVs intracellular processing in hepatocytes and thus the outcome of liver-directed gene therapy using AAV vectors and showed in a proof-of-principle study how this virus-host interaction can be employed to enhance efficacy of this vector system. (Hepatology 2017;66:252-265).

    View details for DOI 10.1002/hep.29176

    View details for PubMedID 28318036

    View details for PubMedCentralID PMC5518300

  • Liver-Mediated Gene Therapy with an Engineered Secretable GAA Transgene Results in Whole-Body Correction of Pompe Disease Ronzitti, G., Colella, P., Puzzo, F., Biferi, M., Bali, D., Paulk, N. K., Vidal, P., Collaud, F., Sola, M., Charles, S., Hardet, R., Leborgne, C., Meliani, A., Cohen-Tannoudji, M., Astord, S., Gjata, B., Sellier, P., van Wittenberghe, L., Vignaud, A., Boisgerault, F., Barkats, M., Laforet, P., Kay, M. A., Koeberl, D. D., Mingozzi, F. CELL PRESS. 2017: 244
  • Long-term exposure to Myozyme results in a decrease of anti-drug antibodies in late-onset Pompe disease patients. Scientific reports Masat, E., Laforêt, P., De Antonio, M., Corre, G., Perniconi, B., Taouagh, N., Mariampillai, K., Amelin, D., Mauhin, W., Hogrel, J. Y., Caillaud, C., Ronzitti, G., Puzzo, F., Kuranda, K., Colella, P., Mallone, R., Benveniste, O., Mingozzi, F. 2016; 6: 36182


    Immunogenicity of recombinant human acid-alpha glucosidase (rhGAA) in enzyme replacement therapy (ERT) is a safety and efficacy concern in the management of late-onset Pompe disease (LOPD). However, long-term effects of ERT on humoral and cellular responses to rhGAA are still poorly understood. To better understand the impact of immunogenicity of rhGAA on the efficacy of ERT, clinical data and blood samples from LOPD patients undergoing ERT for >4 years (n = 28) or untreated (n = 10) were collected and analyzed. In treated LOPD patients, anti-rhGAA antibodies peaked within the first 1000 days of ERT, while long-term exposure to rhGAA resulted in clearance of antibodies with residual production of non-neutralizing IgG. Analysis of  T cell responses to rhGAA showed detectable T cell reactivity only after in vitro restimulation. Upregulation of several cytokines and chemokines was detectable in both treated and untreated LOPD subjects, while IL2 secretion was detectable only in subjects who received ERT. These results indicate that long-term ERT in LOPD patients results in a decrease in antibody titers and residual production of non-inhibitory IgGs. Immune responses to GAA following long-term ERT do not seem to affect efficacy of ERT and are consistent with an immunomodulatory effect possibly mediated by regulatory T cells.

    View details for DOI 10.1038/srep36182

    View details for PubMedID 27812025

    View details for PubMedCentralID PMC5096052

  • The carboxyl-terminal region is NOT essential for secreted and functional levels of coagulation factor X. Journal of thrombosis and haemostasis : JTH Branchini, A., Baroni, M., Burini, F., Puzzo, F., Nicolosi, F., Mari, R., Gemmati, D., Bernardi, F., Pinotti, M. 2015; 13 (8): 1468-74


    The homologous coagulation factor X (FX), VII (FVII), IX (FIX) and protein C (PC) display striking differences in the carboxyl-terminus, with that of FX being the most extended. This region is essential for FVII, FIX and PC secretion.To provide experimental evidence for the role of the FX carboxyl-terminus.Recombinant FX (rFX) variants were expressed in multiple eukaryotic cell systems. Protein and activity levels were evaluated by ELISA, coagulant and amidolytic assays.Expression of a panel of progressively truncated rFX variants in HEK293 cells revealed that the deletion of up to 21 residues in the carboxyl-terminus did not significantly affect secreted protein levels, as confirmed in HepG2 and BHK21 cells. In contrast, chimeric rFX-FVII variants with swapped terminal residues showed severely reduced levels. The truncated rFX variants revealed normal amidolytic activity, suggesting an intact active site. Intriguingly, these variants, which included that resembling the activated FXβ form once cleaved, also displayed remarkable or normal pro-coagulant capacity in PT- and aPTT-based assays. This supports the hypothesis that subjects with nonsense mutations in the FX carboxyl-terminus, so far never identified, would be asymptomatic.For the first time we demonstrate that the FX carboxyl-terminal region downstream of residue K467 is not essential for secretion and provides a modest contribution to pro-coagulant properties. These findings, which might suggest an involvement of the carboxyl-terminal region in the divergence of the homologous FX, FVII, FIX and PC, help to interpret the mutational pattern of FX deficiency.

    View details for DOI 10.1111/jth.13034

    View details for PubMedID 26083275