Ancestral library identifies conserved reprogrammable liver motif on AAV capsid.

Gene therapy is emerging as a modality in 21st-century medicine. Adeno-associated viral (AAV) gene transfer is a leading technology to achieve efficient and durable expression of a therapeutic transgene. However, the structural complexity of the capsid has constrained efforts to engineer the particle toward improved clinical safety and efficacy. Here, we generate a curated library of barcoded AAVs with mutations across a variety of functionally relevant motifs. We then screen this library in vitro and in vivo in mice and nonhuman primates, enabling a broad, multiparametric assessment of every vector within the library. Among the results, we note a single residue that modulates liver transduction across all interrogated models while preserving transduction in heart and skeletal muscles. Moreover, we find that this mutation can be grafted into AAV9 and leads to profound liver detargeting while retaining muscle transduction-a finding potentially relevant to preventing hepatoxicities seen in clinical studies.

Ocular Inflammatory Response to Intravitreal Injection of Adeno-Associated Virus Vector: Relative Contribution of Genome and Capsid.

Both subretinal dosing and intravitreal (IVT) dosing of adeno-associated virus (AAV) in higher species induce mild and transient inflammatory responses that increase with dose. Foreign protein and foreign DNA are known inducers of inflammation, which is also true in the immune-privileged ocular environment. We explored which component(s) of AAV vectors, viral capsid, or viral DNA drive inflammatory responses. Recombinant AAV with three tyrosine to phenylalanine substitutions in the capsid of AAV serotype 2 (rAAV2tYF), and with a generic ubiquitous promoter (cytomegalovirus [CMV]) controlling the expression of humanized green fluorescent protein (hGFP), was processed to enrich for AAV capsids containing genome (full capsids), capsids without genome (empty capsids), and residual material. Nonhuman primate eyes were injected by IVT in both eyes. During in-life, ocular inflammation and development of neutralizing antibodies (NAb) were measured. Following termination, lymph node immunophenotyping was performed, vitreous was processed for cytokine and RNAseq analyses, and ocular sections were assessed for transgene expression (by in situ hybridization) and histopathology. IVT dosing of AAV vectors transiently raised cellular inflammation in the aqueous and induced a more sustained inflammation in the vitreous. Lowering the total capsid dose by removing empty AAV capsids reduced inflammation and improved viral transduction. IVT dosing of AAV induced systemic NAb to AAV irrespective of the vector preparation. Similarly, lymph node immunophenotyping revealed identical profiles irrespective of viral preparation used for dosing. Immune cells in the vitreous were identified based on RNAseq analysis. Three months postdose, cytokine levels were low, indicative of minimal levels of inflammation in agreement with histopathological assessment of the retina.

Evaluating Efficiencies of Dual AAV Approaches for Retinal Targeting.

Retinal gene therapy has come a long way in the last few decades and the development and improvement of new gene delivery technologies has been exponential. The recent promising results from the first clinical trials for inherited retinal degeneration due to mutations in RPE65 have provided a major breakthrough in the field and have helped cement the use of recombinant adeno-associated viruses (AAV) as the major tool for retinal gene supplementation. One of the key problems of AAV however, is its limited capacity for packaging genomic information to a maximum of around 4.8 kb. Previous studies have demonstrated that homologous recombination and/or inverted terminal repeat (ITR) mediated concatemerization of two overlapping AAV vectors can partially overcome the size limitation and help deliver larger transgenes. The aim of this study was to investigate and compare the use of different AAV dual-vector approaches in the mouse retina using a systematic approach comparing efficiencies in vitro and in vivo using a unique oversized reporter construct. We show that the hybrid approach relying on vector genome concatemerization by highly recombinogenic sequences and ITRs sequence overlap offers the best levels of reconstitution both in vitro and in vivo compared to trans-splicing and overlap strategies. Our data also demonstrate that dose and vector serotype do not affect reconstitution efficiency but a discrepancy between mRNA and protein expression data suggests a bottleneck affecting translation.

AAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency Evaluation of AAV Preparations.

Adeno-associated virus (AAV) vectors are promising clinical candidates for therapeutic gene transfer, and a number of AAV-based drugs may emerge on the market over the coming years. To insure the consistency in efficacy and safety of any drug vial that reaches the patient, regulatory agencies require extensive characterization of the final product. Identity is a key characteristic of a therapeutic product, as it ensures its proper labeling and batch-to-batch consistency. Currently, there is no facile, fast, and robust characterization assay enabling to probe the identity of AAV products at the protein level. Here, we investigated whether the thermostability of AAV particles could inform us on the composition of vector preparations. AAV-ID, an assay based on differential scanning fluorimetry (DSF), was evaluated in two AAV research laboratories for specificity, sensitivity, and reproducibility, for six different serotypes (AAV1, 2, 5, 6.2, 8, and 9), using 67 randomly selected AAV preparations. In addition to enabling discrimination of AAV serotypes based on their melting temperatures, the obtained fluorescent fingerprints also provided information on sample homogeneity, particle concentration, and buffer composition. Our data support the use of AAV-ID as a reproducible, fast, and low-cost method to ensure batch-to-batch consistency in manufacturing facilities and academic laboratories.

Targeted inactivation of the mouse epididymal beta-defensin 41 alters sperm flagellar beat pattern and zona pellucida binding.

During epididymal maturation, sperm acquire the ability to swim progressively by interacting with proteins secreted by the epididymal epithelium. Beta-defensin proteins, expressed in the epididymis, continue to regulate sperm motility during capacitation and hyperactivation in the female reproductive tract. We characterized the mouse beta-defensin 41 (DEFB41), by generating a mouse model with iCre recombinase inserted into the first exon of the gene. The homozygous Defb41(iCre/iCre) knock-in mice lacked Defb41 expression and displayed iCre recombinase activity in the principal cells of the proximal epididymis. Heterozygous Defb41(iCre/+) mice can be used to generate epididymis specific conditional knock-out mouse models. Homozygous Defb41(iCre/iCre) sperm displayed a defect in sperm motility with the flagella primarily bending in the pro-hook conformation while capacitated wild-type sperm more often displayed the anti-hook conformation. This led to a reduced straight line motility of Defb41(iCre/iCre) sperm and weaker binding to the oocyte. Thus, DEFB41 is required for proper sperm maturation.

Controlling AAV Tropism in the Nervous System with Natural and Engineered Capsids.

More than one hundred naturally occurring variants of adeno-associated virus (AAV) have been identified, and this library has been further expanded by an array of techniques for modification of the viral capsid. AAV capsid variants possess unique antigenic profiles and demonstrate distinct cellular tropisms driven by differences in receptor binding. AAV capsids can be chemically modified to alter tropism, can be produced as hybrid vectors that combine the properties of multiple serotypes, and can carry peptide insertions that introduce novel receptor-binding activity. Furthermore, directed evolution of shuffled genome libraries can identify engineered variants with unique properties, and rational modification of the viral capsid can alter tropism, reduce blockage by neutralizing antibodies, or enhance transduction efficiency. This large number of AAV variants and engineered capsids provides a varied toolkit for gene delivery to the CNS and retina, with specialized vectors available for many applications, but selecting a capsid variant from the array of available vectors can be difficult. This chapter describes the unique properties of a range of AAV variants and engineered capsids, and provides a guide for selecting the appropriate vector for specific applications in the CNS and retina.

In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector.

Adeno-associated virus (AAV) vectors have emerged as a gene-delivery platform with demonstrated safety and efficacy in a handful of clinical trials for monogenic disorders. However, limitations of the current generation vectors often prevent broader application of AAV gene therapy. Efforts to engineer AAV vectors have been hampered by a limited understanding of the structure-function relationship of the complex multimeric icosahedral architecture of the particle. To develop additional reagents pertinent to further our insight into AAVs, we inferred evolutionary intermediates of the viral capsid using ancestral sequence reconstruction. In-silico-derived sequences were synthesized de novo and characterized for biological properties relevant to clinical applications. This effort led to the generation of nine functional putative ancestral AAVs and the identification of Anc80, the predicted ancestor of the widely studied AAV serotypes 1, 2, 8, and 9, as a highly potent in vivo gene therapy vector for targeting liver, muscle, and retina.

Imbalanced lipid homeostasis in the conditional Dicer1 knockout mouse epididymis causes instability of the sperm membrane.

During epididymal sperm maturation, the lipid content of the sperm membrane is modified, which facilitates sperm motility and fertility. However, little is known about the mechanisms regulating the maturation process. By generating a conditional knockout (cKO) of Dicer1 in the proximal part of the mouse epididymis, we studied the role of RNA interference in epididymal functions. The Dicer1 cKO epididymis displayed an altered lipid homeostasis associated with a 0.6-fold reduction in the expression of the gene elongation of very long chain fatty acids-like 2, an enzyme needed for production of long-chain polyunsaturated fatty acids (PUFAs). Furthermore, the expression of several factors involved in cholesterol synthesis was up-regulated. Accordingly, the Dicer1 cKO sperm membrane showed a 0.7-fold decrease in long-chain PUFAs, whereas the amount of cholesterol in acrosome-reacted sperm displayed a 1.7-fold increase. The increased cholesterol:PUFA ratio of the sperm membrane caused breakage of the neck and acrosome region and immotility of sperm. Dicer1 cKO mice sperm also displayed reduced ability to bind to and fertilize the oocyte in vitro. This study thus shows that Dicer1 is critical for lipid synthesis in the epididymis, which directly affects sperm membrane integrity and male fertility.

Loss of Bmyc results in increased apoptosis associated with upregulation of Myc expression in juvenile murine testis.

Bmyc is a member of the Myc family of transcriptional regulators in the mouse and the rat. It is predominantly expressed in hormonally controlled tissues, with highest level of expression in the epididymis. The BMYC protein has been shown to function as a transcription factor in vitro and to inhibit MYC. To study the significance of BMYC in vivo, a Bmyc knockout (KO) mouse model was generated by homologous recombination. The KO mice were viable and fertile and did not display gross morphological or histological changes compared to the WT mice. However, the testes and the epididymides of the KO mice were smaller than those of the WT mice. Correspondingly, a tendency for a lower sperm concentration in the cauda epididymides of the KO mice was detected. The testosterone produced/testis was significantly reduced, and accordingly, the LH levels were increased in the KO mice. Also, the expression levels of Myc and several of its target genes were elevated in the testes of prepubertal KO mice, whereas no differences in gene expression levels were detected in adult mice. Associated with the increased Myc expression, more apoptotic spermatogenic cells were detected in the seminiferous tubules of the KO mice. In conclusion, our data suggest that Bmyc is a regulator of Myc in vivo and that overexpression of Myc in the developing testis leads to increased apoptosis of spermatogenic cells.

Loss of cysteine-rich secretory protein 4 (Crisp4) leads to deficiency in sperm-zona pellucida interaction in mice.

Mammalian sperm gain their ability to fertilize the egg during transit through the epididymis and by interacting with proteins secreted by the epididymal epithelial cells. Certain members of the CRISP (cysteine-rich secretory protein) family form the major protein constituent of the luminal fluid in the mammalian epididymis. CRISP4 is the newest member of the CRISP family expressed predominantly in the epididymis. Its structure and expression pattern suggest a role in sperm maturation and/or sperm-egg interaction. To study the relevance of CRISP4 in reproduction, we have generated a Crisp4 iCre knock-in mouse model through insertion of the iCre recombinase coding cDNA into the Crisp4 locus. This allows using the mouse line both as a Crisp4 deficient model and as an epididymis-specific iCre-expressing mouse line applicable for the generation of conditional, epididymis-specific knockout mice. We show that the loss of CRISP4 leads to a deficiency of the spermatozoa to undergo progesterone-induced acrosome reaction and to a decreased fertilizing ability of the sperm in the in vitro fertilization conditions, although the mice remain fully fertile in normal mating. However, removal of the egg zona pellucida returned the fertilization potential of the CRISP4-deficient spermatozoa, and accordingly we detected a reduced number of Crisp4-deficient spermatozoa bound to oocytes as compared with the wild-type spermatozoa. We also demonstrate that iCre recombinase is expressed in a pattern similar to endogenous Crisp4 and is able to initiate the recombination event with its target sequences in vivo.

Members of the murine Pate family are predominantly expressed in the epididymis in a segment-specific fashion and regulated by androgens and other testicular factors.

BACKGROUND: Spermatozoa leaving the testis are not able to fertilize the egg in vivo. They must undergo further maturation in the epididymis. Proteins secreted to the epididymal lumen by the epithelial cells interact with the spermatozoa and enable these maturational changes, and are responsible for proper storage conditions before ejaculation. The present study was carried out in order to characterize the expression of a novel Pate (prostate and testis expression) gene family, coding for secreted cysteine-rich proteins, in the epididymis. METHODS: Murine genome databases were searched and sequence comparisons were performed to identify members of the Pate gene family, and their expression profiles in several mouse tissues were characterized by RT-PCR. Alternate transcripts were identified by RT-PCR, sequencing and Northern hybridization. Also, to study the regulation of expression of Pate family genes by the testis, quantitative (q) RT-PCR analyses were performed to compare gene expression levels in the epididymides of intact mice, gonadectomized mice, and gonadectomized mice under testosterone replacement treatment. RESULTS: A revised family tree of Pate genes is presented, including a previously uncharacterized Pate gene named Pate-X, and the data revealed that Acrv1 and Sslp1 should also be considered as members of the Pate family. Alternate splicing was observed for Pate-X, Pate-C and Pate-M. All the Pate genes studied are predominantly expressed in the epididymis, whereas expression in the testis and prostate is notably lower. Loss of androgens and/or testicular luminal factors was observed to affect the epididymal expression of several Pate genes. CONCLUSIONS: We have characterized a gene cluster consisting of at least 14 expressed Pate gene members, including Acrv1, Sslp1 and a previously uncharacterized gene which we named Pate-X. The genes code for putatively secreted, cysteine-rich proteins with a TFP/Ly-6/uPAR domain. Members of the Pate gene cluster characterized are predominantly expressed in the murine epididymis, not in the testis or prostate, and are regulated by testicular factors. Similar proteins are present in venoms of several reptiles, and they are thought to mediate their effects by regulating certain ion channels, and are thus expected to have a clinical relevance in sperm maturation and epididymal infections.

Role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila melanogaster wing imaginal disc.

When a fragment of a Drosophila imaginal disc is cultured in growth permissive conditions, it either regenerates the missing structures or duplicates the pattern present in the fragment. This kind of pattern regulation is known to be epimorphic, i.e. the new pattern is generated by proliferation in a specialized tissue called the blastema. Pattern regulation is accompanied by the healing of the cut surfaces restoring the continuous epithelia. Wound healing has been considered to be the inductive signal to commence regenerative cell divisions. Although the general outlines of the proliferation dynamics in a regenerating imaginal disc blastema have been well studied, little is known about the mechanisms driving cells into the regenerative cell cycles. In this study, we have investigated the role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila wing imaginal disc. By utilizing in vivo and in vitro culturing of incised and fragmented discs, we have been able to visualize the dynamics in cellular architecture and gene expression involved in the healing and regeneration process. Our results directly show that homotypic wound healing is not a prerequisite for regenerative cell divisions. We also show that JNK signaling participates in imaginal disc wound healing and is regulated by the physical dynamics of the process, as well as in recruiting cells into the regenerative cell cycles. A model describing the determination of blastema size is discussed.