Rare monogenic disorders such as lysosomal diseases have been at the forefront in the development of novel treatments where therapeutic options are either limited or unavailable. The increasing number of successful pre-clinical and clinical studies in the last decade demonstrates that gene therapy represents a feasible option to address the unmet medical need of these patients. This article provides a comprehensive overview of the current state of the field, reviewing the most used viral gene delivery vectors in the context of lysosomal storage disorders, a selection of relevant pre-clinical studies and ongoing clinical trials within recent years. Lysosomal storage disorders (LSDs) form a group of genetic errors of metabolism, comprising more than 70 different diseases . These mSpecific treatments that aim to correct the metabolic defect have been developed for many LSDs. These include haematopoietic stem cell transplantation (HSCT), enzyme replacement therapy (ERT), pharmacological chaperone therapy (PCT) and substrate reduction therapy (SRT). HSCT involves the transplantation of haematopoietic stem cells, usually derived from bone marrow. The rationale relies on transplanted stem cells engrafting in the bone marrow and differentiating into various cells of the haematopoietic lineage; some of the cells then cross the blood brain barrier (BBB) and potentially may differentiate into resident microglia of the central nervous system that can secrete functioning lysosomal enzymes ,7. HSCT ®) to CLN2 patients [ERT is based on the premise that LSDs could be treated by administering the recombinant functional enzyme to patients to compensate for their abnormal or defective protein. The concept was originally suggested by Christian de Duve in 1964  and is spatients . The natPCT involves the use of molecular chaperones that may stabilise conformation of unstable or misfolded proteins due to mutations that cause LSDs. This prevents rapid degradation of proteins and allows them to perform their enzymatic function . The onlSRT has been investigated as a potential therapy to treat neurological phenotypes. These small molecules aim to reduce accumulation of undegraded substrate by limiting its synthesis or the synthesis of its precursors ,29. MiglGene therapy is the delivery of nucleic acids to cells for patient benefit using viral or non-viral derived vectors. In the context of LSDs, this is usually delivery of a functional copy of the defective gene. LSDs are an ideal candidate disease group for gene therapy because they are typically well-characterised monogenic disorders for which the defective gene has been identified and animal models have been developed that allow for preclinical testing. For some disorders, the newly synthesized enzyme is secreted into systemic circulation for recapture and use by non-neighbouring cells. This ‘cross-correction’ process allows certain organs, such as the liver, to act as an enzyme factory where the protein is produced and delivered to other organs via blood circulation. Moreover, the amount of enzyme correction required to sufficiently clear the accumulating substrate is estimated to be between 1 and 10% of normal concentrations, although this will vary by disease . Thus, aRetroviruses are lipid-enveloped particles that contain a RNA genome. The viral envelope glycoprotein is responsible for interaction of the retroviral particles with receptors on target cells, thus controlling the host tropism and biodistribution . SubstitLentiviruses are a genus of the retrovirus family. Lentivirus-based vectors mediate more stable transgene expression and efficient gene transfer than previous retroviral vectors . The lenAdenoviruses are non-enveloped viruses with an icosahedral capsid containing a double-stranded DNA (dsDNA) genome . More thAdeno-associated viruses (AAVs) are part of the Parvoviridae family and are replication-deficient non-enveloped viruses. AAVs typically require a helper virus, such as the adenovirus, to facilitate infection. In the absence of a helper virus, the AAV genome remains latent in the host cell  in an epThe theoretical basis of haematopoietic stem cell transplantation for LSDs lies in (1) identification of the M6P receptor pathway , throughIDUA mutations resulting in alpha-L-iduronidase deficiency and affects multiple tissues including the central nervous system (CNS). The first trial was reported in 1981, which described biochemical and clinical improvements of an MPS I-H patient that received allogeneic HSCT at one year of age [The history of HSCT for LSDs started from conventional allogeneic HSCT, which employs HSCs from a healthy person to supply the lysosomal enzyme deficient in the transplant recipient. In the field of LSDs, conventional allogeneic HSCT has performed well in treating MPS I-H, which is caused by r of age . Since tr of age . More rer of age . Encourar of age ,72. A crHaematopoietic stem cell gene therapy (HSC-GT) holds great potential. An advantage of HSC-GT is the ability to perform autologous transplantation, which bypasses the difficulty in donor selection for allogeneic HSCT and avoids the risk of graft-versus-host disease. Addition of gene therapy to HSCT using viral vectors may provide supraphysiological expression of the required lysosomal enzymes as with other gene therapies. This is of particular interest for LSDs, as secretion of the therapeutic protein is enhanced due to gene overexpression in the transplanted cells, resulting in a higher degree of cross-correction. Considering the heredity of all LSDs, matched sibling donors  may also bear the disease mutation and thus cannot provide HSCs of sufficient therapeutic efficacy. The general approach of HSC-GT proceeds as follows: (1) autologous HSCs are collected from the patient, (2) collected HSCs are genetically corrected by transduction with a viral vector harbouring the therapeutic gene and (3) the transduced HSCs are transplanted back into the patient following transplant conditioning treatment similar to that required for HSCT. The most commonly used vectors for HSC-GT are lentiviral and gammaretroviral vectors. Both are able to transduce HSCs as well as to integrate into the host genome, allowing permanent target gene expression by transduced cells and, more importantly, by their progeny in the context of HSCT. However, genome integration is always a double-edged sword with risk of oncogenesis and mutagenesis over time, as discussed above. Thus, gene therapy employing these integrating vectors requires careful safety studies, preferentially with large-scale analysis and long-term follow-up. To date, clinical trials of HSC-GT have been initiated for treatment of various LSDs, including metachromatic leukodystrophy (MLD), MPS IIIA, MPS I-H, cystinosis, Gaucher disease and Fabry disease .ARSA gene. ARSA is a soluble lysosomal enzyme with its lysosomal targeting dependent on the M6P receptor pathway. The physiological properties of ARSA, together with the extensive neurological involvement of MLD, offer hope for the disease to be a potential candidate for HSC-GT. Preclinical HSC-GT studies provided supportive data for clinical trials. Initially, Matzner et al. investigated HSC-GT using a mouse model of MLD and bone-marrow-derived HSCs transduced with a retroviral vector  harbouring the human ARSA gene [ARSA gene, they detected ARSA in both microglia/macrophages in the CNS (representing transduced mononuclear cells engrafted into the CNS) and neurons plus other cell types (providing evidence for cross correction), whereas HSC-GT expressing EGFP resulted in GFP detection only in microglia/macrophages in the CNS [One of the best examples of HSC-GT for treating LSDs, to date, is for metachromatic leukodystrophy. MLD is caused by a deficiency of arylsulfatase A (ARSA) due to mutations in the RSA gene ,74,75. SRSA gene ,77 and aRSA gene  or earlyARSA at pre-symptomatic or very early symptomatic stages, with myeloablative busulfan pre-transplant conditioning. The patients were then followed up for 18–54 months post transplantation. Stable engraftment of transduced HSCs was observed during follow-up, and ARSA activity was reconstituted to physiological or supra-physiological concentrations in haematopoietic cells and in the cerebrospinal fluid (CSF). The therapeutic efficacy was promising. Eight of nine patients showed prevention of disease onset or arrest of progression in accordance with clinical and instrumental assessment. In addition, skin biopsy exhibited occurrence of peripheral nerve remyelination in some of the patients. No serious adverse effects due to the medicinal product were reported during follow-up, and no haematopoietic malignancy was observed. The most frequent adverse events were cytopenia and mucositis, which are common complications of the chemotherapy conditioning for HSCT. A recent case report by Calbi et al. showed post-HSCT endothelial injury could be prevented by using Defibrotide and adjusting busulfan exposure to a lower but still myeloablative area under the curve (AUC) [Based on these preclinical studies, clinical trials have been performed to treat MLD patients. Promising outcomes have been reported from a phase 1/2 clinical trial of HSC-GT in MLD children (NCT01560182) ,79. MLD ve (AUC) , as showN-sulfoglucosamine sulfohydrolase (SGSH) due to mutations in the SGSH gene. Patients usually have onset of disease in their first decade of life and present with heparan sulphate accumulation, progressive neurodegeneration and behavioural abnormalities, leading to death in their second decade. Currently most therapies for MPS IIIA are supportive treatments directed towards multisystem complications, whereas no effective disease-modifying therapy is available to prevent disease progression [SGHG under the CD11b promoter could effectively transduce human HSCs, which could also be effectively scaled up for a sufficient dose necessary for the clinic. HSC-GT with this vector in a humanised mouse model resulted in effective engraftment and biodistribution, with no vector shedding or transmission to germline cells. The vector genotoxicity assay showed low transformation potential, which was comparable to other lentiviral vectors currently used in the clinic. Orchard Therapeutics has initiated a phase 1/2 clinical trial for autologous HSC-GT in MPS IIIA patients using this vector . In summary, compared with other HSC-GT using ubiquitous promoters, this example offers evidence for a strategy using a myeloid-specific promoter to drive transgene expression as an alternative option of (1) boosting expression of the transgene in myeloid cells that engraft and differentiate into microglia-like cells in the CNS, which would be especially useful for LSDs with extensive CNS involvement and (2) preventing potential toxicity associated with supraphysiological enzyme concentrations in the general circulation.Recently, a phase 1/2 clinical trial was initiated (NCT04201405) for treatment of mucopolysaccharidosis type IIIA (MPS IIIA) . MPS IIIgression . HSC-GT gression . The autgression . While igression . The autIDUA gene [IDUA-transduced HSCs were variable between experiments, but a significant reduction of glycosaminoglycan accumulation and histological improvement were consistently observed in visceral organs, with the exception of the brain. A few years later, a study led by Biffi et al. tested HSC-GT mediated by lentiviral vectors and showed complete therapeutic correction in a mouse model [ClinicalTrials.gov Identifier: NCT03488394). In early 2021, Orchard Therapeutics shared the first encouraging preliminary clinical data from the follow up of the initial proof-of concept study, showing sustained IDUA expression in the CSF and normalization of pathology biomarkers in all treated patients [Although allogeneic HSCT has been used as a treatment, HSC-GT has also been studied for MPS I-H. The importance of studying HSC-GT in MPS I-H is to eliminate the problem of histocompatibility in allogeneic HSCT and to potentially provide higher concentrations of the therapeutic enzyme by overexpression for this most severe type of MPS I. An early study using an MPS I-H mouse model firstly evaluated potential therapeutic effects of HSC-GT mediated by an MND retroviral vector expressing the human DUA gene . The tisse model . In partse model ,87,88. Apatients .GBA sequence under the control of the elongation factor 1α short (EFS) promoter. The data on the first treated patient reported a significant decrease in the toxic metabolite Lyso-Gb1 and in the biomarker chitotriosidase, with consequent interruption of ERT [Numerous and successful results in preclinical studies using a type 1 mouse model ,91,92 den of ERT . ImportaCTNS gene encoding the lysosomal membrane-specific transporter for cystine. Cystine accumulation appears in all tissues, and the major cause of death is severe renal damage. A preclinical study by Harrison et al. demonstrated in a Ctns deficient mouse model that HSCs transduced with a lentiviral vector expressing human CTNS were capable of achieving engraftment and expressing transgene in all tissues tested [Ctns deficient mice, serum creatinine, urine phosphate and urine volume were significantly decreased compared with non-treated Ctns deficient mice. To date, a phase 1/2 clinical trial has been initiated to evaluate the efficacy and safety of HSC-GT in cystinosis patients .Although the mechanism of ‘cross-correction’ is applicable for soluble enzymes, diseases caused by defects in transmembrane proteins can also be targeted with an ex vivo gene therapy approach. This is the case for cystinosis, a disease caused by mutations in the s tested . CystineIn addition to MPS IIIA and I-H discussed above, HSC-GT has been studied in animal models for treating other MPS types such as MPS II ,96,97, MGAA gene, resulting in the accumulation of lysosomal glycogen in a variety of cell types. The typical cause of death in this disease is cardio-respiratory failure which results from hypertrophic cardiomyopathy and skeletal muscle weakness. An early study in the Netherlands [GAA reversed cardiac remodelling and improved disease manifestations, without obvious toxicity caused by GAA overexpression. However, complete correction of lysosomal glycogen accumulation in the skeletal muscle and the CNS cells was not achieved, and motor performance was only partially improved. Recently, the same research group conducted an HSC-GT study for Pompe mice using a novel lentiviral vector expressing codon-optimised GAA, which resulted in significantly higher GAA activity in peripheral blood [Numerous pre-clinical studies of HSC-GT have been performed in Pompe disease ,106,107.ApoEII for treating MPS II in animal models, which resulted in the human IDS protein fused to a receptor-binding domain of human ApoE. The theoretical basis of this experiment was the ability of the receptor-binding portion of ApoE to form a high-affinity binding complex with heparan sulphate, which could potentially promote cross-correction in addition to uptake via the M6P-R pathway. The plasma enzyme concentration and uptake across the BBB were elevated, whereas the enzymatic activity was not affected by this modification. When compared to HSCT without vector transduction and HSC-GT with a lentiviral vector expressing normal IDS in an MPS II mouse model, this IDS/ApoEII approach produced significantly enhanced therapeutic effects, resulting in complete correction of brain pathology and behaviour.One of the major advantages of HSC-GT for LSDs compared to conventional HSCT is the ability to overexpress the therapeutic gene in the HSCs and their progeny. This results in higher enzyme concentrations and better cross-correction once the transgene-expressing cells repopulate various tissue compartments. HSC-GT is attractive for treating the neurological manifestation of the diseases compared to other therapies such as ERT that cannot cross the BBB and conventional HSCT which produces relatively lower enzyme levels in the CNS. As discussed above, obtaining sufficient tissue enzyme activity for clinical benefits is an important issue for some LSDs. This has been studied with various optimisiation methods, such as the careful selection of vectors and promoters and codon-optimisation of the gene sequence. There are also alternative approaches that have been studied in the field of LSDs. Gleitz et al.  investigGene editing techniques such as CRISPR-Cas have also been introduced to HSC-GT as an alternative to traditional viral vector-based gene augmentation. With this approach, the addition of the therapeutic gene is achieved by delivering the Cas9 nuclease and a short guide RNA (sgRNA) targeting a nonessential genomic sequence (a “safe harbour”), where an expression cassette (the therapeutic gene with a promoter) will be inserted. In vitro studies have already provided evidence for successful genetic modification of HSCs using CRISPR-Cas9 ,109. VerIn summary, HSC-GT is a valuable gene therapy approach for LSDs. Gene therapy overcomes limitations of allogeneic transplantation and allows amplified expression of the therapeutic gene. Cross-correction via the M6P-R-pathway which is used by many lysosomal proteins, makes it possible to treat disease by correcting a proportion of deficient cells, especially when the therapeutic protein is overexpressed in those cells via gene therapy. In the CNS, HSC-GT is a potentially good option compared to ERT since the recombinant enzyme does not cross the BBB, and to conventional HSCT which shows limited efficacy in the CNS. HSC-GT research is particularly important for LSDs involving extensive pathologies in both the CNS and the periphery, whereas for disease predominantly affecting the brain rather than other organs, direct gene therapy targeting the brain might be more beneficial. Several attempts have been made to improve HSC-GT, as discussed above, and promising results have emerged and paved the way. In the future, several uncertainties or limitations will need to be better addressed: long-term efficacy follow-up and safety analysis, particularly with regard to viral genome integration and transgene overexpression, optimisation of current approaches and technology to better regulate the transgene expression and improve therapeutic effects and reducing complications related to pre-conditioning for transplantation.In vivo gene transfer therapies are based on the ability to introduce the therapeutic genetic material into affected tissues by direct administration into the patient . While eIDUA gene to MPS I mice [GUSB gene [While enzyme replacement therapy is an effective treatment option, systemic gene delivery offers the opportunity for a sustained therapeutic effect comparable to long-term effects of ERT, with a single or limited number of administrations and the additional prospect of treating the CNS. The earliest vectors used for systemic delivery were based on or derived from the retroviral Moloney murine leukaemia virus. These vectors have been used to deliver the S I mice ,113 via USB gene ,115. SimUSB gene . While nUSB gene .GAA [Gammaretroviral vectors have been succeeded by lentiviral vectors based on immunodeficiency viruses and have the ability to also transduce non-dividing cells. Lentiviruses show the ability to efficiently transduce hepatocytes following systemic administration. In fact, gene therapy targeted to the growing liver exploits clonal expansion of the transduced hepatocytes, resulting in high gene expression in the liver. A lentiviral vector based on the feline immunodeficiency virus FIV was first used to deliver the β-glucuronidase gene to MPS VII adult mice with the aim of providing sustained therapeutic enzyme following liver transduction . FurtherGAA . Then, 2GAA  and adulGAA  MPS I knGAA  and IIIAGAA ,124. IntGAA . The autLIPA gene in a mouse model of lysosomal acid lipase (LAL) deficiency [GAA expression in multiple muscle groups in a quail model of Pompe disease [GAA-knock-out mice [Adenoviral vectors have also been employed to deliver the β-glucuronidase gene to MPS VII mice ,127, witficiency . A singl disease  and GAA-out mice ,132. In 13 to 4.5 × 1013 vg/kg) AAV9 gene therapy in infantile (6–12 months) type I and young (2–12 years) type II GM1 gangliosidosis patients (NCT03952637). Despite the challenges associated with manufacturing of the ever larger batches of the viral vector required for intravenous delivery in the clinic, several clinical trials involving adult patients are currently on-going. Adult Fabry disease patients have received AAV6 (NCT04046224) or other engineered capsids  with the aim to test safety and tolerability of the treatment following a single intravenous injection. Similarly, two phase I/II clinical trials delivering AAV8 to adults with late onset Pompe disease , and a self-complementary AAV9 vector to MPSIIA patients (NCT04088734 and long-term follow up NCT02716246) are now active and remain ongoing.With their wide cellular tropism, low toxicity and sustained gene-expression, recombinant AAV vectors represent a compelling candidate for systemic (IV) gene delivery. Although capsids such as AAV8 and AAV9 preferentially transduce liver and muscle , a more 13–3 × 1014 vg/kg) were administered to neonates and children, ranging from elevated serum transaminase in the SMA1 Novartis trial (NCT03306277), complement activation and acute kidney injury in the Duchenne Muscular Dystrophy trial (NCT03362502) to death from sepsis [The ability of AAV vectors, particularly AAV9, to transduce neurons following systemic administration to animal models  and the m sepsis  in the rm sepsis . This imAlthough systemic administration is arguably the most feasible approach to target diseases caused by deficiencies in secreted enzymes, gene delivery has also been used to successfully treat lysosomal defects involving membrane-bound proteins. Neonatal NPC mice received a retro-orbital injection of AAV9 vectors expressing the human NPC1 gene . TreatmeThe selective targeting of organs in order to produce and secrete therapeutic proteins into the bloodstream is an attractive approach to treat systemic diseases such as LSDs. Highly vascularised organs, such as the liver or the lungs, are prime candidates for this gene transfer modality. The development of a successful gene therapy for haemophilia A  and B 1 demonstrGBA vector to cells and mice in a proof-of-concept study, supporting the development of a liver-directed therapy for Gaucher disease [GAA gene under control of a liver-specific promoter are currently being assessed in a clinical trial on late-onset Pompe patients (NCT03533673). A similar approach has been tested in MPS I cats [While the initial choice of serotype was the early characterised AAV2, other capsids such as AAV8, AAV3B and novel engineered vectors including LK03  or NP59  disease . In addi disease ,171, Gau disease  and Pomp disease ,174 mous disease . The safS I cats , and MPSS I cats  and catsS I cats  where geIntraparenchymal administration of an AAV2 vector expressing the murine β-glucuronidase gene to MPS VII mice resulted in increased quantities of enzyme in the viscera as a consequence of both direct transduction of extrahepatic organs and circulation of the enzyme produced in the liver through the bloodstream . InteresIDUA expression in the plasma, completely correcting cardiac, bone, eye and ear manifestations eight months post-administration. Rucker and colleagues raised the idea of early intervention by delivering an AAV vector expressing the human GAA gene to the liver of fetal Pompe mice [For devastating diseases with rapid progression such as most acute forms of LSDs, early intervention is essential to prevent the development of severe pathology and ultimately early death. As already mentioned, viral gene delivery to paediatric patients presents the advantage of preventing or slowing the accumulation of macromolecules at an early stage, when the pathology is not extensive and the progression might still be reversible. Additionally, neonates are more likely to have not yet developed antibodies  or T celmpe mice .Although prenatal and neonatal/paediatric application is an attractive prospect for liver-targeted gene therapy, one has to consider that the fast cell proliferation rate and turnover of hepatocytes can lead to a loss of vector genomes when using vectors considered to be non-integrating such as AAVs . There aThe lung can also be used as a metabolic factory for targeting systemic diseases, exploiting its large surface and extensive vascularisation. In fact, nasal delivery is a non-invasive technique that results in systemic gene expression and might offer the possibility of safe re-administration . An adenSkeletal muscle has also been considered a potential organ factory to produce lysosomal enzymes, especially as certain AAV serotypes are particularly efficient in transducing myofibers . IntramuWhile different gene therapy strategies have been effective in improving the majority of visceral manifestations, the severe neuropathology that characterises many LSDs remains a significant issue for the treatment of these patients. Drug delivery to the central nervous system, particularly to the brain, is challenging because of its unique complexity and limited access through physical barriers such as the skull and the BBB. In the last two decades, AAVs have been the vectors of choice for most of the CNS-directed gene therapy studies .The first approaches were mainly based on the direct infusion of the product into the brain parenchyma. Intraparenchymal (IC) injections have the advantage of providing targeted administration to the affected area and can overcome the challenge of the vector permeating efficiently through the BBB. On the other hand, the limited spread of the vector might not be efficient in treating disorders where the neuropathology is extensive and high concentrations of therapeutic protein expression are required in multiple brain areas. However, this drawback could be overcome by administering the therapy to multiple injection sites in the brain parenchyma, ensuring widespread expression in different regions. This method has been applied in clinical trials for MPS IIIA (NCT01474343), MPS IIIB (ISRCTN19853672), early onset MLD (NCT01801709) and late infantile NCL , where the intervention consisted of six or 12 vector deposits to different brain regions during a single neurosurgical session.CLN6 gene was administered via lumbar puncture to patients with CLN6 type late infantile NCL (NCT02725580), following positive results in NHPs experimentation [Delivery to the cerebrospinal fluid is an alternative approach to achieve widespread gene expression in the CNS. Intrathecal (IT) administration into the lumbar cistern has proven particularly efficient when using AAV9-based vectors . While gentation .IDUA enzyme in the brain, allowing a safe and effective re-administration to the CSF. Together, these studies suggest that CM gene delivery can be a feasible approach for treating the neuropathology in many LSDs, with the advantage of a lower vector dose requirement and reduced exposure of the vector to the peripheral organs compared to systemic administration. In 2020, Prevail Therapeutics started recruiting type II Gaucher patients to enrol in the PROVIDE clinical study (NCT04411654), where infants diagnosed with the acute form of nGD will receive a CM injection of an AAV9 vector. However, it has been shown that CM delivery is not easily translatable to the clinic, as this technique might be associated with risks related to high vascularisation of the tissue in human patients [An extensive study conducted by Ohno et al. compared the kinetics of vector clearance from the CSF following infusion of AAV vectors to the lumbar cistern and the cerebromedullary cistern, or cisterna magna (CM) in NHPs . This anpatients ,255. In patients . The pospatients . This mopatients . The safpatients .Extensive transduction of the CNS is also achieved via intracerebroventricular (ICV) administration. ICV delivery to NHPs results in larger cortical distribution of the vector compared to the intrathecal route, with substantial transduction of the cerebellum  and a coARSA gene was used in a pre-clinical study in metachromatic leukodystrophy [A self-inactivating HIV-based lentivirus vector expressing the human arylsulfatase A ystrophy : 10-montystrophy . This apThe characteristic temporal and spatial expression patterns can limit the applicability of lentiviral vectors for those disorders where widespread transgene expression might not be required or might cause brain toxicity. Widespread gene delivery to the brain has been achieved using a recombinant adenoviral vector expressing the human β-glucuronidase gene to the striatum of MPS VII mice . IncreasSGSH gene [A similar approach was developed for the treatment of MPS IIIA mice, where 6–18-week-old adult mice received bilateral injections into the thalamus with a CAV-2 vector expressing the GSH gene . HoweverDespite CAV-2 vectors having proven effectiveness in transducing neurons following brain-directed administration and their ability to promote widespread gene expression following retrograde axonal transportation , their aWhile brain-directed gene therapy showed promising results for the treatment of the neurological manifestations in many LSDs, correction of the disease is usually limited to the brain. However, other structures of the CNS might be affected by substrate accumulation and cell loss. Batten disease, for example, is characterised by severe and progressive degeneration of the retina . As of tGaa knock-out mice led to glycogen clearance and amelioration of the neuromuscular phenotype. However, prolonged overexpression of GAA is associated with transgene immunogenicity. The immune reaction was successfully attenuated using immune deficient Gaa-KO/SCID mice [Localised administration to the muscle has been widely used to treat skeletal myopathy caused by glycogen accumulation in Pompe disease. Several studies have shown that injection of AAV2 , AAV6 2 and AAV9CID mice . A similGaa knock-out mice [Pompe disease is characterised by weakening of the diaphragm and other respiratory skeletal muscles, resulting in ventilatory insufficiency . Althougout mice . TopicalGaa knock-out mice [GAA and the clearance of glycogen storage in XII motor neurons, with consequent improvement in the upper airway motor function of treated mice.In addition, Pompe patients often present with tongue weakness and typically develop pharyngeal dysphagia due to hypoglossal XII motor neuron dysfunction . In an aout mice . ExploitGaa knock-out mice. Six months post-injection, enzymatic activity was significantly improved in the lungs and myocardium of treated mice, with consequent clearance of glycogen storage. Interestingly, vector genome copies were also detected in the diaphragm and spinal cord, supporting the hypothesis that transduction of respiratory motor neurons can be beneficial in improving respiratory function in Pompe disease.Further research supported the contribution of the CNS to the respiratory muscle impairment in Pompe disease . With thThe gene therapy approaches mentioned so far in this review rely on gene augmentation, rather than correction of the detrimental mutations in the patients’ genome. Genome editing aims to manipulate the patient’s genome in a controlled manner. For example, it may allow insertion of the desired gene at a specific site within the host genome. When targeting actively dividing cells, this would overcome the problems of dilution of a non-integrating vector and the potential genotoxic effects of uncontrolled insertion . The ultGene editing involves the manipulation of the cell system for repairing double-strand breaks in the genome. Two of the most common methods are non-homologous end joining (NHEJ) and homology directed repair (HDR). The NHEJ repair mechanism mediates the direct re-ligation of the broken DNA without the need of a homologous template to guide repair. The NHEJ mechanism is the quickest at restoring the DNA strand; however, the repair mechanism is highly error prone and sequence independent, frequently leading to small insertions and deletions (indels) . This caHDR is a naturally occurring system which can repair DNA DSBs with high efficiency. HDR requires a homologous DNA repair template, which contains the desired edit within stretches of the homologous sequence both immediately upstream and downstream of the target site. These are typically ~1.2 kb in length for mammalian cells ,283,284.Zinc finger nucleases (ZFNs) are synthetic, engineered proteins consisting of the non-specific cleavage domain of FokI endonuclease and zinc finger proteins . These m11 vg of both AAV8-ZFNs and 1.5 × 1012 vg AAV8-donors which contained either acid-β-glucosidase (Gaucher), α-galactosidase A (Fabry), α-L-iduronidase (MPS I) or iduronate-2 sulfatase (MPS II). The ZFNs were designed to induce a double-strand break in the albumin gene, while the donor genes were flanked by homologous repeats and a splice acceptor sequence. Four weeks post-administration, supraphysiological concentrations of enzymes were detected in the liver, while a 3-fold increase in enzymatic activity persisted for eight weeks post injection. Sequencing analysis of the genomic DNA showed a 31.4% modification at the intended target with less than 2% at queried off target loci.In the LSD field, approaches with ZFNs have been explored for Gaucher disease , Fabry dIDUA knockout mouse models, aged 4 to 10 weeks old, were intravenously injected with 1.5 × 1011 vg of each AAV8-ZFN targeted at intron 1 of the albumin locus and 1.2 × 1012 vg AAV8-IDUA donor [IDUA activity in the liver was 10- to 16-fold higher compared to wild-type controls, and enzyme activity in the plasma was 7- to 9-fold higher compared to the control mice at 28 days post injection. Secondary tissues including the spleen, heart, lungs and muscle also showed an increase in IDUA activity one month post-administration; however, no significant increase was observed in the brain. This suggested that uptake by the M6P-R pathway allowed for a degree of cross correction to occur, as confirmed by the reduction of the glycosaminoglycan concentrations in the liver and all peripheral tissues at the four-month time point.α-L-iduronidase UA donor . This ap11 vg AAV-ZFN and 1.2 × 1012 vg AAV-donor. The enzyme concentration in the liver was 207-fold higher compared to the wild-type mice at four months post-injection, with the GAG concentration restored to wild-type levels.Similar experiments were also performed on a mouse model of MPS II . Male miIDUA activity, the change of urine GAG levels and AAV6 clearance. The trial sponsored by Sangamo Therapeutics has been terminated as the study did not demonstrate a clear clinical benefit.In a clinical trial for the treatment of MPS I (NCT02702115), three participants were enrolled in a phase 1/2 trial to receive ascending single doses of SB-318  administered by intravenous infusion. The purpose of this study was to assess the safety and tolerability of the agent, with the secondary outcomes being measured including the change in Xanthomonas [The transcription activator-like effector (TALE) protein is derived from the pathogenic plant bacteria thomonas ,293. Thethomonas ,295. Exothomonas ,296.A Nobel Prize winning advancement in the field of gene editing was the discovery of the CRISPR system . BacteriIDUA gene into the albumin locus [IDUA cDNA [IDUA sequence. At 11 months post-administration, a significant increase in IDUA activity was observed in the liver, heart, spleen and brain tissue of the animals treated with the highest dose; 3 × 1014 vg/kg AAV-IDUA and 5 × 1013 vg/kg AAV-Cas9. These encouraging results were also reflected in the GAG concentrations with a significant decrease in the treated mice compared to the untreated controls. Examination of the tissue by light microscopy revealed a reduced incidence of foam cells in the liver and decreased vacuolation of Purkinje cells in the brain of treated mice. Noticeable behavioural improvement was observed, with treated mice showing restored memory and learning ability.The use of CRISPR in the field of LSDs has been explored for the creation of novel disease models  and the in locus . A systeDUA cDNA . NeonataHEXM), which incorporates the active site of subunit-α and stable subunit-β interface along with areas from each subunit required to interact with the GM2 activator protein. Together, these subunits form a homodimer capable of degrading GM2 gangliosides [9 vg/g) and AAV8-HEXM-sgRNA (3 × 1010 vg/g) via the temporal facial vein. One month post administration, the plasma 4-methylumbelliferyl-β-N-acetylglucosamine-6-sulphate (MUGS) and 4-methylumbelliferyl-β-N-acetylglucosamine (MUG) activity increased 144-fold and 17-fold, respectively, when compared to the wild-type. The tissues were harvested following euthanasia at four months of age and assessed for enzymatic activity. There was a 7-fold increase in the liver of treated mice compared to the wild-type, and a significant increase in the brain, heart and spleen compared to the untreated group. The rotarod results showed that there was also a significant locomotor improvement in the treated mice.Another similar approach using the albumin locus as a safe harbour has also been investigated for the treatment of Tay-Sachs and Sandhoff diseases . The insliosides ,301,302.IDUA to allow for HDR. The agents were first delivered to human fibroblasts using a non-viral nano-emulsion system, and then to new-born MPS I mice via systemic administration. The serum IDUA activity was restored to around 6% of normal, and the enzymatic activity in the peripheral organs examined showed a significant increase compared to the untreated controls.A frequent mutation in South American MPS I patients is a 1205G>A mutation which leads to the formation of a premature stop codon ,303. TheCCR5 gene and Cas9 protein were electroporated into HPSCs followed by AAV6-mediated transduction for delivery of the homologous templates [CCR5 site was chosen as the safe harbour, because it is a non-essential gene which poses no detrimental effect at deficiency. The frequency of modification was reported as 54 ± 10% for cord blood-derived HSPCs, and 44 ± 7% for peripheral blood-derived HSPCs. The IDUA gene was placed under the control of the spleen focus forming virus (SFFV) or phosphoglycerate kinase promoters, which led to a 250-fold and 50-fold increase in enzymatic activity, respectively. Upon co-culture with MPS I patient-derived fibroblasts, the modified HSPCs led to a decrease in lysosomal compartment size by means of cross correction. Subsequently, homozygous NOD-scid-gamma (NSD)-IDUAX/X mice were created by CRISPR-Cas9 as a model of MPS I. At 18 weeks post-engraftment, the GAG concentrations in urine had reduced by 65% compared to the sham-treated mice.Ex vivo gene editing has also been evaluated for the treatment of MPS I ,304. In emplates . The CCRCCR5 gene was selected as the safe harbour, and the glucocerebrosidase expression cassette was inserted at the targeted site. The modified HSCs were confirmed for their capacity for long-term engraftment and multi-lineage differentiation in the injected mice, as well as confined glucocerebrosidase overexpression specific to the monocyte/macrophage lineage.The research group then established this approach for potential treatment of another LSD; Gaucher disease . Again, A further iteration of the CRISPR/Cas9 system has been the development of base editors, which come in two classes: cytosine base editors (CBEs) , which aOne of the factors which has limited the use of base editors for in vivo work so far, has been the size of the DNA strand encoding the editor . At 5.2 10 vg of each AAV. The treatment led to a 9.2% increase in lifespan compared to the untreated mice. At around 100 days post-injection, the number of surviving Purkinje neurons was found to have modestly increased in the treated mice to 38% of wild-type levels. In order to examine the extent of editing efficiency, the brain tissue was also examined, with nearly 50% of the cortical neurons been edited.This system was then explored for its therapeutic potential for the treatment of Niemann-Pick type C disease . The T31Base editors are a powerful tool in the genome editing toolkit; however, they are limited to transition purine-to-purine or pyrimidine-to-pyrimidine mutations. Prime editing is a novel technology capable of making a wider range of changes to the genome such as targeted insertions, targeted deletions, all four transition mutations and all eight transversion mutations, either alone or in combination . Prime eHEXA gene at the 1278 position [HEXA gene of HEK293T cells, with a 31% efficiency and 0.8% indel rate. The authors then isolated two cell lines that were homologous for the mutated gene and attempted to rectify the mutation. The most successful correction to wild-type HEXA had 33% efficiency and 0.32% indel formation [With regards to LSDs, the most common mutation which causes Tay-Sachs disease is a TATC insertion into the position . Anzalonormation . These rLysosomal diseases have proven to be fertile ground for the development of gene therapy. This is understandable given the number of conditions, knowledge of the defective gene, the availability of animal models for pre-clinical studies and the frequent absence of effective treatments. Some of these conditions may be considered ‘lower hanging fruit’, where the gene therapy approach would benefit from secretion of the protein or enzyme from transduced cells and uptake by other untransduced cells. However, improvement in the viral vector technology and a better understanding of the appropriate route of administration now permit targeting of more difficult conditions. These involve non-secreted or membrane-bound proteins where efficacy is more dependent on the transduction efficiency of the viral vector in the relevant organs. Engineering of the vector and expression cassette can improve transduction efficiency and penetration into specific tissues. This is particularly important for LSDs with a neurological component, where efficient administration of the vector to the CNS is essential and may also require supplementation via other routes of delivery.While gene therapy is an attractive therapeutic option, ethical concerns related to the possible risk profile require careful consideration. The absence of alternative treatments is clearly a strong argument in favour of gene therapy; however, the risk of extending the life span of the patient while only partially restoring their pathology without significant improvements in their quality of life, is still a critical concern. Therefore, scientists and clinicians should be mindful and carefully evaluate suitability of the therapy to protect both patients and caregivers’ interests.Although LSDs are rare disorders, the large number of gene therapy companies invested in the LSD field is encouraging and also gives a sense of the optimism for future licensed therapies for conditions with unmet medical needs. In addition, progress in developing novel therapies proceeds in parallel with the assessment of new biomarkers necessary to diagnose new patients earlier and more efficiently and to monitor the course of the treatments over time. Overall, this advancement means that the outcome of the ongoing and soon to be initiated clinical trials is of vital importance, first to the patients and their families, but also for future development of novel advanced therapy medicinal products in other fields other than gene augmentation therapy for LSDs.


Epigenetic memory plays a critical role in the establishment and maintenance of cell identities in multicellular organisms. Polycomb and trithorax group (PcG and TrxG) proteins are responsible for epigenetic memory, and in flies, they are recruited to specialized DNA regulatory elements termed polycomb response elements (PREs). Previous transgene studies have shown that PREs can silence reporter genes outside of their normal context, often by pairing sensitive (PSS) mechanism; however, their silencing activity is non-autonomous and depends upon the surrounding chromatin context. It is not known why PRE activity depends on the local environment or what outside factors can induce silencing.Drosophila, we find that the so-called neutral chromatin environments vary substantially in their ability to support the silencing activity of the well-characterized bxdPRE. In refractory chromosomal contexts, factors required for PcG-silencing are unable to gain access to the PRE. Silencing activity can be rescued by linking the bxdPRE to a boundary element (insulator). When placed next to the PRE, the boundaries induce an alteration in chromatin structure enabling factors critical for PcG silencing to gain access to the bxdPRE. When placed at a distance from the bxdPRE, boundaries induce PSS by bringing the bxdPREs on each homolog in close proximity.Using an attP system in This proof-of-concept study demonstrates that the repressing activity of PREs can be induced or enhanced by nearby boundary elements.The online version contains supplementary material available at 10.1186/s12915-021-01047-8. Drosophila embryo by the three bithorax complex (BX-C) homeotic genes: Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) [Ubx determines segment identity in two parasegments, PS5 and PS6, and its expression in these parasegments is controlled by the abx/bx and bxd/pbx regulatory domains, respectively. abx/bx is activated at the blastoderm stage in PS5, while bxd/pbx is silenced. In PS6, both domains are active, but Ubx expression is controlled by bxd/pbx.In many developmental systems, the mechanisms involved in the choice of cell fate are distinct from those deployed for maintaining or remembering cell fate decisions. One classic example of choice is the establishment of parasegment identity in the posterior 2/3rds of  (Abd-B) –3. Theseon or off during the rest of development. The memory mechanism is epigenetic. The off or silenced state is remembered by Polycomb group proteins (PcG), while the on or active state is maintained by trithorax group proteins (TrxG) [The maternal and early zygotic gene products that initially determine the appropriate activity state of the BX-C regulatory domains for each parasegment disappear after the onset of gastrulation. Consequently, a different mechanism is required to remember whether the domain is s (TrxG) –10. PcG s (TrxG) –16.Drosophila, most of the known PcG proteins are components of three complexes: polycomb repressive complexes 1 and 2 (PRC1 and PRC2) and Pho repressive complex (PhoRC). The canonical PRC1 core subunits are sex combs extra , posterior sex combs (Psc), polycomb (Pc), and polyhomeotic (Ph) proteins [In proteins –19. The proteins , 21. Finproteins  and DNA-proteins . The Trxproteins , 25.abx/bx and bxd/pbx, that control Ubx expression [engrailed and even-skipped [. The binding of PcG/TrxG proteins to PREs is mediated by sequence-specific DNA-binding proteins, including Pho [The PcG and TrxG proteins are recruited to their regulatory targets by specialized DNA elements called PREs (polycomb response elements) –29. PREspression , 31. The-skipped , 33. Theding Pho , 34 and ding Pho .white reporter and a PRE were compared in hemizygotes and homozygotes. In classical PSS, there is little or no evidence of silencing in hemizygous flies, while strong silencing is evident when the transgene is homozygous. Since PSS is typically observed for transgenes inserted at the same chromosomal site, but not at different sites, the enhancement of repression in homozygotes is thought to be due to trans interactions between the two copies of the PRE, one on each homolog [One characteristic activity of PREs that can be readily assayed is their ability to induce pairing-sensitive silencing (PSS) , 36. Thi homolog , 36. The homolog .Abd-B region of BX-C, the PREs for the iab-5, iab-6, iab-7, and iab-8 regulatory domains are located in relatively close proximity to the chromatin boundary elements (insulators) that flank these domains [Mcp for iab-5; Fab-6 for iab-6; Fab-7 for iab-7, and Fab-8 for iab-8 [Fab-7 and Mcp, the PRE and boundary are integral parts of the same regulatory element [Mcp boundaries were shown to facilitate PSS between transgene inserts megabases from each other and even between transgenes inserted on different chromosomes [The activity of PRE elements in these transgene experiments is influenced by the surrounding chromatin environment. Depending on the integration site, PREs can repress and have a neutral or even a positive impact on gene transcription , 38–46.  domains , 47–49. or iab-8 . Moreove element , 50–52. omosomes –56. Simiomosomes .cis and the other in trans appear to be in play. The first is observed when the boundary is placed in close proximity to the PRE. In this case, silencing is activated in fly hemizygous for the transgene insert and is enhanced when the transgene is homozygous. The induction of silencing activity in hemizygotes is not dependent on the identity of the linked boundary and is observed for all three boundaries. ChIP experiments indicate that the linked boundary facilitates alterations in the local chromatin structure. In the region spanning the PRE, there is a depletion of histone H3 levels and a concomitant recruitment of factors required for PRE function. Interestingly, in the region to either side of the boundary, PRE sequences histone H3 levels are increased. The second mechanism is observed when the boundary element is separated by 1 kb or more from the PRE. In this case, silencing is not or at most only weakly activated in hemizygotes. However, silencing activity is induced in homozygous flies. In this case, the mechanism is dependent upon boundary:boundary pairing interactions as it is not observed when the PREs are linked to heterologous instead of homologous boundaries.In the present study, we tested whether a close linkage between boundaries and PREs can impact the acquisition of silencing activity. We show that in chromosomal contexts that are refractory to PRE-induced silencing, the PREs fail to recruit PcG/TrxG proteins. However, both silencing and recruitment of PRE-associated proteins can be triggered by linking the PRE to an artificial boundary element consisting of multimerized binding sites for the Su(Hw), CTCF, or Pita polydactyl zinc finger DNA-binding proteins. Two different mechanisms, one that occurs in Drosophila transgenic lines. For this purpose, we selected five attP sites that had previously been shown to provide a context in which the expression of a white reporter is not subject to obvious repression or activation by the surrounding chromatin neighborhood . The bxdPRE was flanked by ~1 kb “neutral” spacers derived from coding regions of the eGFP and RFP genes and by terminators of transcription (SV40 terminator upstream and yellow gene terminator downstream) to reduce the influence of potential transcription from surrounding genomic sequences. To better assess the effects of the PRE, we also inserted a control construct at each site, which has all of these components except for bxdPRE  with antibodies against Ph, which is a core component of the PRC1 complex  that is known to be enriched in PcG/TrxG proteins. Figure bxdPRE in the four lines that show silencing of the white gene. Moreover, the extent of association correlates well with the level of repression observed in each line. Consistent with the lack of silencing of white in the 96E insertion, Ph is not found to be associated with its bxdPRE sequence.Silencing is expected to be accompanied by the association of PcG proteins with the Abd-B regulatory domains are positioned close to the boundary elements for each domain. Another example is one of the PREs for the even-skipped (eve) gene that is located next to the distal boundary of the eve locus homie [PREs are often located near other transcriptional regulatory elements. As noted above, the PREs in the four us homie . These obxdPRE is unable to silence white at this insertion site. As the test boundary, we used an artificial element consisting of multimerized binding sites for the polydactyl zinc finger protein Su(Hw) rather than an endogenous boundary. Endogenous boundaries contain binding sites for many different proteins, and some are known to be required for PRE activity. For example, the GAF protein is implicated not only in insulation but also in PcG-dependent silencing [To explore this possibility, we selected the 96E attP since the ilencing .gypsy transposon [gypsy transposon has 12 binding sites for the Su(Hw) protein [gypsy insulator is sufficient for boundary activity in transgene reporter assays [bxdPRE  multimer would not be able to insulate white from PRE-dependent silencing [bxdPRE has a dramatic effect on white expression. Silencing of white is evident in hemizygotes, while strong PSS is observed when the transgene is homozygous. In contrast, the 4xSu(Hw) multimer alone has no effect on eye pigmentation either as a hemizygote or a homozygote. Thus, the presence of the 4xSu(Hw) multimer can induce the establishment of silencing by the bxdPRE in a chromosomal location that is not conducive to PcG-dependent silencing.The Su(Hw) protein is responsible for the boundary activity of the insulator element associated with the ansposon , 67. The protein , 69; howr assays  and in tr assays . This 4xilencing , 57, 72.bxdPRE insertions that are able to repress white expression but is not found associated with the bxdPRE at 96E. If the addition of the 4xSu(Hw) multimer induces PcG-dependent repression, it should also facilitate the recruitment of Ph to the 96E bxdPRE. To test this prediction, we used ChIP to examine Ph association at 5 sites in the Su-bxd and bxd transgenes: (1) the distal end of the spacer sequence upstream of bxdPRE, (2) bxdPRE, (3) the distal end of the spacer sequence downstream of bxdPRE, (4) the white transcription unit, and (5) the white promoter  associates with the bxdPRE element in the transgene construct, but not with other sequences even though white expression is silenced [bxdPRE and the 4xSu(Hw) multimer as Ph is not associated with the control transgene carrying only the 4xSu(Hw) multimer  multimer is included in the transgene, but not when it is absent. The DNA-binding proteins known or thought to be important for bxdPRE silencing include Pho, which binds PREs together with its partner Sfmbt (the PhoRC complex) as well as the Combgap DNA-binding protein. An alternative model is that the bxdPRE at 96E is unable to silence because it is occupied by TrxG proteins and these factors block association of Ph and other PcG proteins and/or their function. In this case, the presence of the 4xSu(Hw) multimer could shift the balance to favor of the recruitment of PcG complexes.Biochemical and genetic studies have shown that, like other PREs, the silencing activity of the bxdPRE in the Su-bxd transgene. These results are consistent with the predictions of the first model. We infer from these finding that the presence of the 4xSu(Hw) multimer induces the association of key DNA-binding proteins with the bxdPRE.To test these two models, we used antibodies against Pho, Sfmbt, Combgap, and two TrxG proteins, Trx and CBP, for ChIP experiments. In the bxd transgene, we observe only background levels of Pho, Sfmbt, and Combgap in ChIPs for the PRE and other sequences in the transgene Fig. c–e. In cbxdPRE alone, but will be displaced by PcG proteins when the 4xSu(Hw) multimer is present. However, like the PcG proteins, the Trx and CBP are recruited to the bxdPRE only when the 4xSu(Hw) multimer is present  protein. For this reason, we asked whether the 4xSu(Hw) multimer is able to augment bxdPRE silencing at the four other attP sites which do not have peaks for Su(Hw) nearby  multimer  is included next to bxdPRE in the transgene  multimer is present  peak that overlaps the attP site at 96E, while there are two larger peaks on either side of the attP ~5 kb and ~10 kb away , both of these multimers have insulating activity in BX-C boundary replacement experiments [bxdPRE induces silencing activity in hemizygotes and PSS in homozygotes. Control experiments show that white expression is not affected when the multimers are included in the transgene alone. Thus, multimerized sites for three different chromosomal architectural proteins can induce the silencing activity of the bxdPRE.Next, we wondered whether induction of PRE repressing activity is unique to Su(Hw) or whether other polydactyl zinc finger proteins that have chromosome architectural functions are able to induce PRE silencing. To test this, we linked the eriments , 75–77. enPRE (PSE2) from the engrailed locus, in different chromosomal environments. In previous P-element transgene experiments, this element was shown to repress white expression as a hemizygote and/or homozygote and maintain the parasegmental expression pattern of a Ubx reporter [white by the enPRE in hemizygote flies is weak or nonexistent at all five attP insertion sites and there is no evidence of PSS when the inserts are homozygous   multimer to the Hw) Fig. c.bxdPRE is increased. In the latter model, relatively small changes in distance (<10 kb) should have little or no effect on the ability of the boundary to target the transgene to a PcG-silenced compartment.How do the architectural proteins help establish PRE activity? Two different mechanisms could be in play. The architectural proteins could facilitate the establishment of PcG silencing by locally displacing nucleosomes and/or by recruiting chromatin remodeling complexes so that key DNA-binding factors and PcG complexes are able to assemble on the PRE. Alternatively, the architectural proteins could enhance PRE activity by helping target the transgene from an active chromosomal compartment to a PcG-silenced chromosomal compartment . Since tbxdPRE by 1 kb and 3 kb . A 1-kb distance would be sufficient for approximately five nucleosomes, while 3 kb would correspond to about fifteen nucleosomes. All of the constructs were then introduced into the 96E attP site. The results of this analysis are shown in Fig. bxdPRE adversely impacts silencing activity. For the 4xSu(Hw) multimer, a distance of 1 kb is sufficient to substantially reduce silencing . In the case of the 4xCTCF multimer, the disruption of silencing is greater when it is located 3 kb away from the bxdPRE than it is at a 1-kb distance; however, even at 1 kb, silencing is reduced compared to the control CTCF-bxd construct. These findings argue in favor of the first model, namely that the 4xSu(Hw) and 4xCTCF multimers act in cis to facilitate the assembly of silencing complexes on the PRE  or 4xCTCF multimers and the  kb Fig. a. For thbxdPRE, this is not true for PSS. As shown in Fig. bxdPRE represses white expression even when the boundary multimers are located 3 kb away. A plausible explanation for this result is that pairing of the 4xSu(Hw) or 4xCTCF multimers in trans would tend to stabilize pairing interactions between the PREs on each homolog, and this interaction facilitates the PSS-dependent assembly of functional silencing complexes.While silencing activity in hemizygotes is significantly compromised when the multimers are moved away from the Drosophila depends upon specific interactions between proteins associated with each element [trans to CTCF-1kb-bxd or Su-3kb-bxd trans to CTCF-3kb-bxd. Figure trans and is not observed in heterologous combinations. A possible model is shown in Fig. Boundary pairing in  element , 84–86.  element , 87, 88. element . On othe element . Thus, ibxdPRE for silencing in hemizygotes and enhanced PSS in homozygotes suggests that the boundary has a local effect on chromatin structure that enables PcG factors to gain access to the PRE and assemble functional silencing complexes. If this suggestion is correct, then the chromatin organization of the bxdPRE should be altered when it is closely linked to a boundary element. To test this prediction, we analyzed histone H3 association with six unique sequences (1n-6n) located at different distances upstream and downstream of the BamH1 site used to insert test DNAs  has no apparent effect on histone H3 occupancy and the profile across the six sequences is similar to that of the E-w control. A different result is obtained for Su, CTCF, or Pita transgenes containing the multimerized binding sites for the boundary proteins. In all three cases, there is a reduction in histone H3 occupancy in the sequences located immediately next to the multimers  and is equivalent to that observed for the control genomic sequence in the coding region of the Ras64B gene (ras) Fig. b. The iners Fig. b. This fbxdPRE combination is reduced. In each case, the reduction is approximately the same as that observed for the corresponding multimer alone. Importantly, this is true for 4n, which is separated from the multimers by the 650 bp bxdPRE. These results provide strong support for the idea that closely linked boundary elements can induce alterations in the association of nucleosomes with the PRE. Interestingly, this is not the only alteration in nucleosome association evident in these transgenes. In all three of the multimer-bxdPRE combinations, we found that histone H3 association with the more distant sequences  is enhanced relative to the various control transgenes . Since enhanced association is not observed with the inactive bxdPRE or with any of the multimers alone, it seems likely that this effect is due to the recruitment of functional PcG complexes to the bxdPRE. It remains to be determined whether this alteration in histone H3 association reflects an increase in nucleosome density in the flanking DNA regions or an increase in the extent of compaction.We next examined the Su-bxd, CTCF-bxd, and Pita-bxd transgenes Fig. b. As wascis and requires a close linkage of the boundary and PRE. This mechanism locally alters the pattern of histone association and facilitates the recruitment of factors critical for PcG repression. The other takes place in trans and is mediated by boundary:boundary pairing interactions. In this second mechanism, boundaries appear to provide a “spot weld” that holds the homologs in close proximity. This facilitates PRE:PRE interactions and results in PSS even when the boundary multimers are separated from the PREs. We undertook several additional experiments to further explore this “pairing” mechanism.The studies in the previous sections indicate that boundaries can enhance PRE silencing by two different mechanisms. One takes place in Boundary pairing can induce silencing in trans: When 4xSu(Hw) or 4xCTCF is placed next to the bxdPRE, the PRE can assemble a functional silencing complex and repress white in hemizygotes. However, the bxdPRE would not be expected to efficiently silence white in trans unless the homologs are tightly paired in the immediate neighborhood. To test this expectation, we generated trans-heterozygotes between the starting transgene E-w  and either Su-bxd or CTCF-bxd , CTCF, or Pita-binding sites. Figure white in the three trans-heterologous combinations of multimerized binding sites is close to that observed when both transgenes have multimerized binding sites for the same protein.An inactive PRE can be transactivated by an active PRE: The bxdPRE insertion in the 22A attP site is a classic example of PSS. Little or no silencing is observed in hemizygotes, while silencing is quite efficient in homozygotes. The PSS phenomenon suggests that PRE:PRE interactions in trans can synergizes, promoting the assembly of functional PcG silencing complexes on both PREs. To test this idea for 96E, we generated trans combinations between the inactive PRE in the bxd transgene and transgenes in which bxdPRE is activated by closely linked 4xSu(Hw), 4xCTCF, or 5xPita sites. Consistent with prediction, the white reporter in the homolog carrying the bxdPRE (only) transgene is repressed when the other homolog has the boundary-bxdPRE , strong silencing of white is observed in hemizygotes, while at the other extreme (96E) there is no evidence of silencing even in homozygotes. For the three remaining attP sites, there is a range of silencing in hemizygous and homozygotes. For 22A and 58A, there is little evidence of silencing in hemizygous flies, while PSS is observed in homozygotes. In the case of 51C, white expression is clearly repressed in hemizygotes, and repression increases in homozygotes. The strength of silencing at the five attP sites correlates well with the recruitment of the PcG protein Ph. High levels are found associated with bxdPRE when it is inserted at 68E, while only background levels are evident at 96E.PcG-dependent silencing is widely used during the development of multicellular organisms as a mechanism for maintaining the determined state –10. In fbxdPRE insert at 96E.These findings are consistent with previous P-element transgene-based experiments, which showed that the silencing activity of PREs is non-autonomous and depends upon the local chromosomal context , 44–46. bxdPRE. We found that all three of these artificial boundaries were able to induce silencing in hemizygotes and PSS in homozygotes. For the 4xSu(Hw)-bxdPRE combination, we found that the acquisition of silencing activity is accompanied by the recruitment of both components of the PhoRC DNA binding complex, Pho and Sfmbt, and other PcG silencing factors to the bxdPRE. Surprisingly, we found that two TrxG proteins, Trx and CBP, are also associated with the bxdPRE, and like the PcG factors, this association is only observed when the bxdPRE is closely linked to the 4xSu(Hw) multimer.These findings led us to wonder what features of the chromatin landscape in the neighborhood of the PRE might facilitate the recruitment of the required silencing factors. In this respect, it was of interest that PREs in BX-C are often closely linked to chromatin boundary elements . This cobxdPRE. Induction of silencing is distance dependent, and for the 4xSu(Hw) multimer, a distance of 1 kb is sufficient to largely eliminate silencing in hemizygotes, while it is reduced but not entirely eliminated for the 4xCTCF multimer. The importance of proximity taken together with the de novo recruitment of DNA binding and PcG proteins suggests that the boundary might alter the local chromatin structure so that factors important for PcG-dependent silencing can gain access to the PRE. Consistent with this hypothesis, we find that multimerized binding sites for the boundary factors Su(Hw), CTCF, and Pita can, on their own, generate a local reduction in histone H3 occupancy, indicating that the multimers likely form a nucleosome-free region of chromatin like that observed for many endogenous boundaries [bxdPRE is placed next to these multimers, the nucleosome free region extends across the 650-bp bxdPRE fragment. While we did not investigate the mechanisms of nucleosome displacement, Su(Hw) and CTCF have been shown to interact with chromatin remodeling complexes. Su(Hw) was found to recruit the Brahma chromatin remodeling protein [In hemizygotes, silencing activity is induced when the boundary multimers are placed next to the undaries –92. When protein , 94. Mor protein . It woulbxdPRE, the presence of multimers in the transgene even at a distance of 3 kb is sufficient to promote PSS when the transgene is homozygous. Critically, the induction of PSS in this configuration is dependent on the ability of the boundaries on each homolog to pair with each other as it is not evident when the 4xSu(Hw) multimer is trans to the 4xCTCF multimer. A plausible interpretation of this finding is that when inactive PREs on each homolog are brought into close proximity by the paired boundaries, there is a synergistic interaction, which is able to promote the acquisition of PcG-dependent silencing activity. This model predicts that an active PRE should be able to trans-activate an inactive PRE. Consistent with this prediction, we found that an active bxdPRE (linked to a multimer) on one homolog can partially trans-activate an inactive bxdPRE (unlinked to a multimer) on the other homolog.Consistent with idea that the boundary multimers facilitate the displacement of nucleosomes from the PRE, we find that silencing activity is compromised when the multimers are placed at a distance of 1 to 3 kb from the PRE. This distance effect would seem to argue against a mechanism in which the multimers target the PRE to a PcG compartment. On the other hand, while the boundary multimers are unable to fully augment silencing activity in hemizygotes unless they are next to the bxdPRE, the artificial boundaries we tested are able to autonomously establish regions of chromatin that have reduced nucleosome occupancy. When the bxdPRE is placed next to one of these multimers, the region of reduced nucleosome occupancy extends over the PRE. Accompany this reduction in occupancy, PcG and TrxG factors are found associated with the bxdPRE and silencing activity is observed. In addition to this local effect on PcG silencing activity, we found that boundaries can induce PSS when placed at a distance from the PRE. In this case, our genetic experiments indicate that boundary:boundary pairing forms a “spot weld” which enables the PREs on each homolog to interact with each other and induce PcG silencing.Here, we show that boundary elements can help induce the silencing activity of PREs by facilitating the recruitment of DNA-binding and PcG/TrxG proteins. Unlike the yacw1118 flies, and the transgenic progeny was identified by their eye pigmentation. Details of crosses used for genetic analysis are given in Additional file All flies were maintained at 25°C on the standard yeast medium. The constructs were injected into embryos of lines ZH-attP-22A BDSC #24481), ZH-attP-51C (BDSC #24482), ZH-attP-58A (BDSC #24484), ZH-attP-68E (BDSC #24485), and ZH-attP-96E (BDSC #24487) . The res481, ZH-awhite expression, we visually determined the degree of pigmentation in the eyes (white) of 3- to 5-day-old males developing at 25°C, with reference to standard color scales. Pigmentation of all flies was analyzed in hemi- (P/+) or homozygotes (P/P).For phenotype analysis of BamHI-EcoRI fragment containing the white gene (mini-white version) without Wari insulator was clones from pCaSpeR∆700 vector [white gene (attBdir-white-rev-pSK). The sequence corresponding to the eye enhancer (E) of the white gene (regulatory sequences from position –1180 to –1849 bp relative to the transcription start site) was cut out of the Ee− pBluescript SK+ plasmid [white gene (attBdir-white-rev-E-rev-pSK).The 0 vector  into the plasmid  without yellow gene terminators of transcription were cloned downstream eGFP and Cherry respectively in orientation to stop the transcription from flanking regions (SV40dir-eGFPrev-Cherrydir-Ytermrev).The coding regions of eGFP (PCR-amplified with primers 5′-atggtgagcaagggcgaggagct-3′ and 5′-cttgtacagctcgtccatgccga-3′) and Cherry (PCR-amplified with primers 5′-atggtgagcaagggcgaggag-3 and 5′-ttacttgtacagctcgtccat-3′) were cloned into pBluescript SK+ vector in a “head-to-head” orientation (eGFPrev-Cherry-pSK). The SV40 and white-rev-E-rev-pSK plasmid between attB and the white gene. The resulting E-w construct has BamHI cloning site between the eGFP and RFP coding regions that was used to create the following constructs. The 656bp bxdPRE (corresponding to the genome sequences between PstI-NdeI) was PCR amplified from the frt(PRE) plasmid [BglII, XhoI, and SpeI restriction sites) and reverse primer . The bxdPRE PCR fragment was then cleaved by BamHI-BglII and cloned into E-w cleaved by BamHI (bxd construct). The 4xSu(Hw) sites (4xSu) were cut from a plasmid used previously [I and SpeI (Su-bxd construct) or into the E-w construct cleaved by BamHI (Su construct). CTCF-bxd, CTCF, Pita-bxd, Pita, en, and Su-en constructs were generated by the same scheme using the following plasmids and fragments. The plasmids with 4xCTCF [I restriction site was inserted upstream of eGFP (SphI-bxd). The 4xSu fragment or 4xCTCF was inserted into SphI-bxd cleaved by SphI. To create Su-3kb-bxd, CTCF-3kb-bxd constructs, the 2087-bp fragment containing the LacZ coding region was inserted into Su-1kb-bxd, CTCF-1kb-bxd constructs between 4xSu and eGFP. The sequences corresponding to construct fragments  are given in Additional file To obtain E-w construct, the fragment corresponding to SV40dir-eGFPrev-Cherrydir-Ytermrev was cloned into attBdir- plasmid  with direviously  and clonh 4xCTCF  and 5xPih 4xCTCF  sites weN-lauroylsarcosine, EDTA-free protease inhibitor cocktail) and sonicated to break chromatin into fragments with an average length of 700 bp. The material was pelleted by centrifugation at 18 000 g for 5 min, and the supernatant fluid was transferred to a new tube. The pellet was treated with the second 0.5-ml portion of lysis buffer, and the preparation was centrifuged at 18 000g for 5 min. The two portions of the supernatant fluid were pooled, cleared by centrifuging twice at 18 000g for 10 min, and the resultant chromatin extract (1 ml) was used for ChIP experiments after preincubation with A-Sepharose or G-Sepharose. One aliquot (1/10 volume) of chromatin extract after preincubation with Sepharose was kept as a control sample (Input).For each experiment, 150–200 mg of heads from 2- to 5-day-old adults homozygous for the different constructs were collected. Experimental procedures for chromatin immunoprecipitations were performed as described previously , 63. BriChIP experiments involved incubation with specific rabbit/rat antibodies or with nonimmune IgG that was used as nonspecific antibody control. All ChIP experiments were made at least in triplicate. The enrichment of specific DNA fragments was analyzed by real-time qPCR, using a C1000™ Thermal Cycler with CFX96 real-time PCR detection module (Bio-Rad) or a StepOne Plus Thermal Cycler . Primers used in ChIP/real-time PCR analyses are listed in Additional file Escherichia coli, affinity purified on Ni Sepharose 6 Fast Flow  according to the manufacturer’s protocol and injected into rabbits following the standard immunization procedure. Antibodies were affinity-purified from serum on the same antigen as was used for immunization. The specificity of Combgap, CBP, and Pho antibodies was verified by RNAi/Western blotting , Sfmbt (1-348 aa of isoform PB) , 63, andAdditional file 1: Table S1. Eye phenotypes of hemizygote transgene flies with insertion of white marker gene without enhancer at selected attP lines. Figure S1. X-ChIP for the “Su” transgene with Ph antibody and X-ChIP for the “Su-bxd” and “Su” transgenes with Su(Hw) antibody. Table S2. Primers used for X-ChIP-qPCR analysis to test PcG/TrxG, DNA-binding proteins recruitment to transgenes. Table S3. Primers used for X-ChIP-qPCR analysis to test histone H3 binding to transgenes. Figure S2. Antibody specificity test.Additional file 2. This file contains the analysis of homologue chromosomes contact frequencies at the 22A, 51C, 58A, 68E and 96E attP insertion sites.Additional file 3. This file contains the ChIP-seq data for PcG and boundary proteins binding to 22A, 51C, 58A, 68E and 96E genome regions.Additional file 4 This file contains: (1) the DNA sequences of the construct elements. (2) the details of Drosophila genetic crosses.


This cohort study evaluates the association of the COVID-19 pandemic with gender differences in work-family factors and mental health among physician parents. Has the COVID-19 pandemic been associated with differences in careers and mental health between physician mothers and fathers?In this cohort study of 276 physicians during the COVID-19 pandemic, mothers were more likely than fathers to be responsible for childcare or schooling and household tasks, to work primarily from home, to reduce their work hours, and to experience work-to-family conflict, family-to-work conflict, and depressive and anxiety symptoms. A gender difference in depressive symptoms was observed among physician parents during the COVID-19 pandemic that was not present before the pandemic.This study suggests that pandemic conditions are associated with an increase in gender inequalities within medicine and signals the importance of further attention and resources to mitigate the potential adverse consequences for the careers and well-being of physician mothers. The COVID-19 pandemic has placed increased strain on health care workers and disrupted childcare and schooling arrangements in unprecedented ways. As substantial gender inequalities existed in medicine before the pandemic, physician mothers may be at particular risk for adverse professional and psychological consequences.To assess gender differences in work-family factors and mental health among physician parents during the COVID-19 pandemic.This prospective cohort study included 276 US physicians enrolled in the Intern Health Study since their first year of residency training. Physicians who had participated in the primary study as interns during the 2007 to 2008 and 2008 to 2009 academic years and opted into a secondary longitudinal follow-up study were invited to complete an online survey in August 2018 and August 2020.Work-family experience included 3 single-item questions and the Work and Family Conflict Scale, and mental health symptoms included the Patient Health Questionnaire–9 (PHQ-9) and Generalized Anxiety Disorder–7 scale.The primary outcomes were work-to-family and family-to-work conflict and depressive symptoms and anxiety symptoms during August 2020. Depressive symptoms between 2018 (before the COVID-19 pandemic) and 2020 (during the COVID-19 pandemic) were compared by gender.P < .001) and household tasks  during the pandemic compared with men. Women were also more likely than men to work primarily from home  and reduce their work hours . Women experienced greater work-to-family conflict , family-to-work conflict , and depressive  and anxiety  symptoms compared with men. We observed a difference between women and men in depressive symptoms during the COVID-19 pandemic  that was not present before the pandemic .Among 215 physician parents who completed the August 2020 survey, 114 (53.0%) were female and the weighted mean (SD) age was 40.1 (3.57) years. Among physician parents, women were more likely to be responsible for childcare or schooling (24.6%  vs 0.8% ; This study found significant gender disparities in work and family experiences and mental health symptoms among physician parents during the COVID-19 pandemic, which may translate to increased risk for suicide, medical errors, and lower quality of patient care for physician mothers. Institutional and public policy solutions are needed to mitigate the potential adverse consequences for women’s careers and well-being. For physicians and their families, the increased strain on the US health care system and prolonged childcare and schooling disruptions have created unprecedented professional and personal challenges. Physicians and medical professional organizations have described heightened struggles to balance work and family responsibilities under pandemic conditions and have voiced concern over the potential harmful psychological and career consequences, particularly for female physicians.3 However, data on the gendered implications of the pandemic among physicians based on validated scientific measures remain scarce.4With more than 100 million cases and 3 million deaths globally,5 and compensation.6 An emerging body of literature suggests that gender disparities in work and family roles and conflict between those roles may be an important factor,10 particularly for women in dual-physician families.12 Pandemic changes in mental and physical demands at work and at home may exacerbate these existing gender inequalities and may therefore pose a greater threat to the career progression of female physicians, especially mothers. At the same time, as frontline health care workers during an infectious disease outbreak, physicians face unique stressors that place them at greater risk for worsening mental health.15 As work-family conflict was previously shown to be associated with depression,16 burnout,17 and emotional exhaustion18 for female physicians, mothers might be at particular risk for experiencing adverse psychological effects with increased pandemic work and family stress.Even before the COVID-19 pandemic, female physicians faced considerable barriers to achieving gender parity in career advancement20 and recent cross-sectional studies suggest high levels of job concern21 and anxiety among physician mothers,22 to our knowledge, the differential effect of the COVID-19 pandemic on psychological well-being has not yet been evaluated longitudinally among physician parents. Given the preexisting gender disparities within medicine and the marked increase in household and caregiving responsibilities under pandemic conditions, investigation of the gendered work-family and mental health experiences of physicians during this period and the ways in which existing drivers of inequity may be exacerbated during such crises is critical to understand potential implications for gender equity within medicine and inform intervention efforts for the current situation as well as mitigation strategies for the future.23 The current study sought to address this knowledge gap by assessing work-family factors and mental health symptoms among physician mothers and fathers during the COVID-19 pandemic using survey data collected from a national longitudinal cohort of early-career US physicians.Although early evidence points to the presence of a gender gap in research productivity among academics in general24 Physicians who had participated in the primary study as interns during the 2007 to 2008 and 2008 to 2009 academic years and opted into a secondary longitudinal follow-up study were invited to complete an online survey in August 2018 and August 2020. All participants provided informed consent via the website and were compensated $25. The University of Michigan institutional review board approved the study. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelineThis cohort study included US physicians enrolled in the Intern Health Study, a prospective cohort study assessing stress and depression during the first year of residency training in the US composed of roughly equal proportions of women and men.Participants reported general demographic and work information, including partner’s occupation and employment status. To evaluate work and family experiences during the COVID-19 pandemic, respondents were asked to indicate whether (1) they experienced a loss of childcare or a school closure, (2) they or their partner worked primarily from home, or (3) they or their partner voluntarily reduced their work hours. Participants were also asked who was primarily responsible for (1) childcare or schooling and (2) day-to-day household tasks.25 Respondents rated their level of agreement with each item on a 7-point Likert scale from 1 (very strongly disagree) to 7 (very strongly agree). Scores range from 5 to 35 points. The Work and Family Conflict Scale has strong concurrent validity and demonstrates high internal consistency (>.90) and reliability.To evaluate the extent that family interfered with work roles and responsibilities (family-to-work conflict) and work interfered with family roles and responsibilities (work-to-family conflict), participants were asked to complete the Work and Family Conflict Scale.26 Individuals rated items on a 4-point Likert scale from 0  to 3 (nearly every day). Total scores range from 0 to 27. A score of 10 or greater on the PHQ-9 has a sensitivity of 88% and a specificity of 88% for the diagnosis of major depression.To assess depressive symptoms, participants were asked to complete the Patient Health Questionnaire–9 (PHQ-9).27 Participants rated items on a 4-point Likert scale from 0  to 3 (nearly every day). Total scores range from 0 to 21. The GAD-7 has 89% sensitivity and 82% specificity for detecting clinical generalized anxiety disorder.To assess anxiety symptoms, participants were asked to complete the Generalized Anxiety Disorder 7-item scale (GAD-7).28 To account for differences between participants who completed the 2020 annual survey and those who did not, we used variables correlated with survey completion to calculate probability of participation using the PSMATCH SAS procedure  with variables correlated with the completion. The attrition weights (w2) for each person who completed the 2020 annual survey was the inverse of the probability of participation. The final weights were the product of poststratification weights and attrition weights (w1b × w1w × w2).We used a sample weighting strategy to reduce the potential bias introduced from differences between participants and nonparticipants and between participants who completed the annual 2020 survey and those who did not. To reduce potential bias owing to sampling in estimating the changes over time in all factors of interest, we used data from the Association of American Medical Colleges (AAMC) on the overall characteristics of first-year residents in the US in 2007 and 2008 as the reference target and performed a 2-step poststratification ranking on the study data to construct weights such that the distribution of cohort year, sex, specialty, and race and ethnicity in the sample matched the AAMC data distribution. The first step was to generate between-cohort weights (w1b) with the raking variable to be cohort year; the second step was to generate weights within each cohort (w1w) with the raking variables to be sex, specialty, and race and ethnicity. The poststratification rankings were performed with the R package anesrake .2 tests. To assess the association of different family circumstances with the potential to affect parental burden, we conducted sequential analyses on increasingly restricted samples. First, we assessed the sample of physicians who had at least 1 child younger than 18 years and compared female and male physicians without any children as a control. Then, we restricted the analysis to the subset of physician parents who were working full-time before the COVID-19 pandemic with a full-time physician partner. To account for potential differences in household and childcare responsibilities based on child age, we included the age of the youngest child (<6 years vs ≥6 years) as a covariate in the analysis. We constructed 95% CIs for proportions from binary data using the exact binomial test. To adjust for multiple comparisons, the significance level of 2-sided α < .05 was corrected according to the Bonferroni procedure.All analyses were restricted to physicians who had completed their medical training. To compare work and family experiences between physician parents by gender, we conducted a series of weighted χTo identify factors associated with work-family conflict and anxiety and depressive symptoms, we constructed a series of multivariable weighted regression models with the following participant characteristics: gender, age of the youngest child, number of children, sleep hours during the past week, weekly work hours during the past month, partner employment status, partner occupation, and specialty. Categorical variables were modeled as indicator variables with a reference category.t test, limiting the analysis to participants who were parents in both years (n = 180). All analyses were performed using SAS, version 9.4. Two-sided P values <.05 were considered statistically significant.To assess whether gender differences in depressive symptoms emerged among physician parents during the pandemic, we compared gender differences in PHQ-9 scores between 2018 (before the COVID-19 pandemic) and 2020 (during the COVID-19 pandemic) using the weighted 29), work full time before the COVID-19 pandemic, and have a partner who did not currently work full time  participant age was 40.1 (3.57), and 114 participants (53.0%) were women. Men were more likely than women to be married, have 3 or more children, work in a surgical specialty  but did not differ significantly from nonrespondents by gender or specialty. Of the participants, 4 (1.4%) had not completed their medical training, and 57 (20.7%) did not currently have a child younger than 18 years and were therefore excluded from the analysis.In 2007 and 2008, 740 of 1271 invited interns (58.2%) enrolled in the primary study. Of these participants, 507 of 740 (68.5%) opted into an additional longitudinal follow-up arm. Between 2009 and 2020, 80 participants (15.8%) were lost to follow up. In total, 276 of 427 contacted physicians (65.1%) completed the August 2020 survey. Compared with all participants in 2007 and 2008, respondents in 2020 were slightly younger  (P < .001). Women were also significantly more likely to perform the majority of day-to-day household tasks than were men . Among physicians working full-time with full-time physician partners, the gender differences in household responsibilities  and childcare and schooling  were even greater than in the overall samples.After accounting for sampling weight and the age of the youngest child, among physician parents, women were significantly more likely to lose childcare during the COVID-19 pandemic compared with men  . Among wP < .001) and to voluntarily reduce their work hours  during the pandemic compared with men. Women with full-time physician partners were even more likely to primarily work from home  and reduce their work hours  than were men with full-time physician partners. No significant gender differences in having a work from home partner or partners reducing work hours were observed.Women were twice as likely to primarily work from home  and family-to-work  conflict (P = .01).In a weighted model accounting for specialty, number of children, age of the youngest child, and partner’s employment status, mothers reported greater work-to-family  and GAD-7 anxiety symptom scores  (P = .003) and GAD-7 score .In a weighted model accounting for specialty, number of children, age of the youngest child, and partner’s employment status, mothers reported greater PHQ-9 depressive symptoms  . Fewer sP = .009). In contrast, there was no statistically significant gender difference in mean (SD) PHQ-9 scores found in this cohort in 2018  . Consistent with the full weighted sample, mean (SD) PHQ-9 depressive symptom scores in 2020 were significantly higher for women compared with men  . Among iFindings from this national cohort study of early-career US physicians suggest that the COVID-19 pandemic is associated with an increase in existing work-family balance struggles for physician mothers, adversely affecting their professional lives and psychological well-being. Of note, we observed a significant gender difference in depression that was not present before the COVID-19 pandemic.33 Consistent with other professions, female physicians tend to perform a greater share of domestic and caregiving duties than male physicians and are 3 times as likely to sacrifice work time to care for their children.34 During the COVID-19 pandemic, we identified an even greater disparity, with physician mothers 30 times more likely to manage childcare and schooling than physician fathers. Among dual-physician couples, none of the men reported taking a primary role in managing the increased pandemic caregiving demands.Parenthood has previously been associated with reduced work hours, income, and retention for women in medicine, with female physicians more likely to be part of a dual-career family and to experience greater work-family balance challenges.35 in line with recent trends in other fields,36 we found that physician mothers disproportionately downshifted at work by reducing their work hours or working primarily from home. Consistent with prior work on dual-physician marriages,12 we found that women with physician partners were even more likely to make these adjustments during the pandemic.With their greater overall pandemic workload, we found that women experienced significantly higher levels of conflict between their roles as physicians and parents. To relieve the intensifying pressures of their professional life and “second shift” at home,18 burnout,17 and depression16 among female physicians. Under increased pandemic strain, we identified a similar pattern in which physician mothers’ risk for depression increased significantly compared with physician fathers’. Of note, no gender difference in depression risk was present in this group before the COVID-19 pandemic. Although high rates of anxiety have been identified among physician mothers during the pandemic,22 this study is the first, to our knowledge, to demonstrate the differential and longitudinal association of the pandemic with the mental health of physician mothers. As physician depression has been associated with increased risk for suicide, medical errors, and lower quality of patient care,39 this has important implications for the well-being of physician mothers as well as their patients.Work-family conflict has been associated with emotional exhaustion,40 and lower productivity.19 Even short-term adjustments can have serious long-term repercussions as they may lead to lower earnings and negatively impact opportunities for promotion,41 further exacerbating gender inequalities in compensation6 and advancement.4 Although the greater availability of telehealth services has increased work flexibility,42 women who are rarely onsite may miss out on critical career opportunities and experience the added strain of simultaneously managing work and childcare at home.Given existing gender inequities in medicine, the career stakes of the COVID-19 pandemic for physician mothers are high. Early evidence from the pandemic suggests that women experience more negative professional consequences than men under work-from-home conditions10 As some recent studies have found that female physicians spend more time with patients43 and have better patient outcomes,44 this loss could be devastating to the US health care system, particularly in the context of a global pandemic and an impending physician shortage. For those physician mothers forced out of the workforce, creating a viable path for re-entry into medicine after the pandemic will be critical.With the increased burden placed on physician mothers during the pandemic, our findings suggest that the existing gender gap in early-career physician retention, which are primarily associated with family factors, will continue to grow.46 Increased support for family care needs, including childcare and paid family leave, as well as wellness programs tailored for physician mothers are vital to protect against adverse effects on productivity and well-being during the pandemic and beyond.47 Initiatives to increase work schedule and location flexibility are also critical.45 In addition, as the stark gender differences in pandemic experiences identified among dual-physician couples have shown, a shift in the culture around work and family balance in medicine is needed. Efforts to normalize the use of parental leave and sick days among male physicians, for example, could help challenge existing gender biases in work and family expectations. An initiative similar to #TimesUp in health care that aims to increase awareness and address inequities related to work-family challenges may be critical to improving workplace conditions and mental health for women.48Further institutional and public policy solutions are needed to mitigate the potential adverse effects of the COVID-19 pandemic for women’s careers and well-being and to ensure that recent gender equity gains in medicine are not lost. Given the current gender gaps in pay and promotion, institutions should actively work to recruit, retain, and advance women and be vigilant that cost-cutting measures and career advancement metrics do not disproportionately penalize them.This study has strengths. We used detailed measures and scientifically validated tools to assess gender differences in work-family conflict and mental health during the COVID-19 pandemic. Furthermore, our longitudinal study design facilitated the comparison of depressive symptoms among the same participants before and during the COVID-19 pandemic, which allowed us to assess whether a gender gap in depressive symptoms emerged during the pandemic that was not identified in prior work, to our knowledge. We also minimized the risk of response bias present in many COVID-19 studies by including the COVID-19 experience questions as part of a routine follow-up survey in an existing physician cohort.This study also has limitations. First, the sample consisted of early-career physicians in the US; thus, the results may not be generalizable to all physicians or to physicians in other countries. Second, although the sample weighting can help to mitigate bias, definitive causal conclusions cannot be drawn from observational studies. It is possible other factors related to participant attrition may have influenced the study results, although we found no meaningful differences in age, gender, or specialty between respondents and those lost to follow-up. Third, because of the limited sample size, the precise magnitude estimates of the group differences identified in this study were not possible. Fourth, all data were obtained via self-report rather than a diagnostic interview, and longitudinal data were not available for family responsibilities, work and family conflict, and anxiety symptoms. Future investigation of the role of other factors, such as sexual orientation, race, ethnicity, immigration status, relationship status, geographical location, and income as well as other personal  and work factors  would be beneficial.This cohort study is, to our knowledge, the first to systematically examine gender differences in work-family conflict and well-being in the same cohort of physician parents before and during the COVID-19 pandemic. Findings from this national study suggest that gender disparities within medicine may have increased in association with pandemic work and home conditions, with disproportionate consequences for the mental health and careers of physician mothers. These findings underscore the importance of taking immediate action to ensure women have access to the resources and supports necessary to navigate this unprecedented and uncertain time and the work-family challenges that may ensue moving forward.