In vivo genome editing using novel AAV-PHP variants rescues motor function deficits and extends survival in a SOD1-ALS mouse model.

CRISPR-based gene editing technology represents a promising approach to deliver therapies for inherited disorders, including amyotrophic lateral sclerosis (ALS). Toxic gain-of-function superoxide dismutase 1 (SOD1) mutations are responsible for ~20% of familial ALS cases. Thus, current clinical strategies to treat SOD1-ALS are designed to lower SOD1 levels. Here, we utilized AAV-PHP.B variants to deliver CRISPR-Cas9 guide RNAs designed to disrupt the human SOD1 (huSOD1) transgene in SOD1G93A mice. A one-time intracerebroventricular injection of AAV.PHP.B-huSOD1-sgRNA into neonatal H11Cas9 SOD1G93A mice caused robust and sustained mutant huSOD1 protein reduction in the cortex and spinal cord, and restored motor function. Neonatal treatment also reduced spinal motor neuron loss, denervation at neuromuscular junction (NMJ) and muscle atrophy, diminished axonal damage and preserved compound muscle action potential throughout the lifespan of treated mice. SOD1G93A treated mice achieved significant disease-free survival, extending lifespan by more than 110 days. Importantly, a one-time intrathecal or intravenous injection of AAV.PHP.eB-huSOD1-sgRNA in adult H11Cas9 SOD1G93A mice, immediately before symptom onset, also extended lifespan by at least 170 days. We observed substantial protection against disease progression, demonstrating the utility of our CRISPR editing preclinical approach for target evaluation. Our approach uncovered key parameters (e.g., AAV capsid, Cas9 expression) that resulted in improved efficacy compared to similar approaches and can also serve to accelerate drug target validation.

Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice.

CRISPR-Cas systems have emerged as a powerful tool to generate genetic models for studying normal and diseased central nervous system (CNS). Targeted gene disruption at specific loci has been demonstrated successfully in non-dividing neurons. Despite its simplicity, high specificity and low cost, the efficiency of CRISPR-mediated knockout in vivo can be substantially impacted by many parameters. Here, we used CRISPR-Cas9 to disrupt the neuronal-specific gene, NeuN, and optimized key parameters to achieve effective gene knockout broadly in the CNS in postnatal mice. Three cell lines and two primary neuron cultures were used to validate the disruption of NeuN by single-guide RNAs (sgRNA) harboring distinct spacers and scaffold sequences. This triage identified an optimal sgRNA design with the highest NeuN disruption in in vitro and in vivo systems. To enhance CRISPR efficiency, AAV-PHP.B, a vector with superior neuronal transduction, was used to deliver this sgRNA in Cas9 mice via neonatal intracerebroventricular (ICV) injection. This approach resulted in 99.4% biallelic indels rate in the transduced cells, leading to greater than 70% reduction of total NeuN proteins in the cortex, hippocampus and spinal cord. This work contributes to the optimization of CRISPR-mediated knockout and will be beneficial for fundamental and preclinical research.

Telomere dysfunction induces metabolic and mitochondrial compromise.

Telomere dysfunction activates p53-mediated cellular growth arrest, senescence and apoptosis to drive progressive atrophy and functional decline in high-turnover tissues. The broader adverse impact of telomere dysfunction across many tissues including more quiescent systems prompted transcriptomic network analyses to identify common mechanisms operative in haematopoietic stem cells, heart and liver. These unbiased studies revealed profound repression of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha and beta (PGC-1α and PGC-1ß, also known as Ppargc1a and Ppargc1b, respectively) and the downstream network in mice null for either telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. Consistent with PGCs as master regulators of mitochondrial physiology and metabolism, telomere dysfunction is associated with impaired mitochondrial biogenesis and function, decreased gluconeogenesis, cardiomyopathy, and increased reactive oxygen species. In the setting of telomere dysfunction, enforced Tert or PGC-1α expression or germline deletion of p53 (also known as Trp53) substantially restores PGC network expression, mitochondrial respiration, cardiac function and gluconeogenesis. We demonstrate that telomere dysfunction activates p53 which in turn binds and represses PGC-1α and PGC-1ß promoters, thereby forging a direct link between telomere and mitochondrial biology. We propose that this telomere-p53-PGC axis contributes to organ and metabolic failure and to diminishing organismal fitness in the setting of telomere dysfunction.

Maintenance and repair of the lung endothelium does not involve contributions from marrow-derived endothelial precursor cells.

Lung endothelium is believed to be a quiescent tissue with the potential to exhibit rapid and effective repair after injury. Endothelial progenitor cells derived from the bone marrow have been proposed as one source of new endothelial cells that may directly contribute to pulmonary endothelial cell homeostasis and repair. Here we use bone marrow transplantation models, using purified hematopoietic stem cells (HSCs) or unfractionated whole marrow, to assess engraftment of cells in the endothelium of a variety of tissues. We find scant evidence for any contribution of bone marrow-derived cells to the pulmonary endothelium in the steady state or after recovery from hyperoxia-induced endothelial injury. Although a rare population of CD45-/CD31+/VECadherin+ bone marrow-derived cells, originating from HSCs, can be found in lung tissue after transplantation, these cells are not readily found in anatomic locations that define the pulmonary endothelium. Moreover, by tracking transplanted bone marrow cells obtained from donor transgenic mice containing endothelial lineage-selective reporters (Tie2-GFP), no contribution of bone marrow-derived cells to the adult lung, liver, pancreas, heart, and kidney endothelium can be detected, even after prolonged follow-up periods of 11 months or after recovery from hyperoxic pulmonary endothelial injury. Our findings argue against any significant engraftment of bone marrow-derived cells in the pulmonary vascular endothelium.

The in vivo genetic program of murine primordial lung epithelial progenitors.

Multipotent Nkx2-1-positive lung epithelial primordial progenitors of the foregut endoderm are thought to be the developmental precursors to all adult lung epithelial lineages. However, little is known about the global transcriptomic programs or gene networks that regulate these gateway progenitors in vivo. Here we use bulk RNA-sequencing to describe the unique genetic program of in vivo murine lung primordial progenitors and computationally identify signaling pathways, such as Wnt and Tgf-ß superfamily pathways, that are involved in their cell-fate determination from pre-specified embryonic foregut. We integrate this information in computational models to generate in vitro engineered lung primordial progenitors from mouse pluripotent stem cells, improving the fidelity of the resulting cells through unbiased, easy-to-interpret similarity scores and modulation of cell culture conditions, including substratum elastic modulus and extracellular matrix composition. The methodology proposed here can have wide applicability to the in vitro derivation of bona fide tissue progenitors of all germ layers.

Endothelial protein C receptor (CD201) explicitly identifies hematopoietic stem cells in murine bone marrow.

The hematopoietic stem cell (HSC) is a unique cell type found in bone marrow, which has the capacity for both self-renewal and differentiation into all blood lineages. The identification of genes expressed specifically in HSCs may help identify gene products vital to the control of self-renewal and/or differentiation, as well as antigens capable of forming the basis for improved methods of stem cell isolation. In previous studies, we identified a number of genes that appeared to be differentially expressed in murine bone marrow-derived HSCs, using microarray technology. We report here that one of those genes, encoding the murine endothelial protein C receptor (EPCR), is expressed at high levels within the bone marrow in HSCs. Bone marrow cells isolated on the basis of EPCR expression alone are highly enriched for hematopoietic reconstitution activity, showing levels of engraftment in vivo comparable to that of stem cells purified using the most effective conventional methods. Moreover, evaluation of cell populations first enriched for stem cell activity by conventional methods and subsequently fractionated on the basis of EPCR expression indicates that stem cell activity is always associated with EPCR-expressing cells. Based on our findings, we believe EPCR represents the first known marker that 'explicitly' identifies hematopoietic stem cells within murine bone marrow.

Complete correction of murine Artemis immunodeficiency by lentiviral vector-mediated gene transfer.

Artemis gene mutations are responsible for the development of a severe combined immunodeficiency [radiation-sensitive (RS) SCID] characterized by a severe B and T cell deficiency and a normal natural killer cell population. To establish the feasibility of a gene therapy approach to the treatment of RS-SCID, we generated a series of lentiviral vectors expressing human Artemis from different promoters and used them to transduce highly purified hematopoietic stem cells (HSCs) from Artemis knockout mice. HSCs transduced by the different viruses were transplanted into either lethally irradiated Rag-1-deficient animals or Artemis knockout mice treated with a nonmyeloablative dose of Busulfan. In both models, transplantation of HSCs transduced by a vector that used a murine phosphoglycerate kinase (PGK) promoter led to a complete functional correction of the immunodeficiency. Corrected animals displayed rescue of mature B cells with normal levels of serum immunoglobulins, together with complete rescue of the T cell compartment as evidenced by the presence of mature T lymphocytes in peripheral blood as well as normal values of thymocytes in thymus. Those B and T cells were capable of activation, as shown both by in vitro stimulation responses and in vivo after immune challenge. Overall, the results indicate that a gene therapy approach for RS-SCID involving the transplantation of genetically modified HSCs is indeed feasible. Furthermore, our studies suggest the possibility that nonmyeloablative conditioning regimens might be effectively used to promote engraftment of genetically modified cells in the case of diseases where standard irradiation-based myeloablative bone marrow transplantation protocols may prove problematic.

A novel stem-cell population in adult liver with potent hematopoietic-reconstitution activity.

A number of recent reports have documented that cells possessing hematopoietic-reconstitution ability can be identified and isolated from a variety of solid organs in the adult animal. In all studies to date, however, purified organ-derived stem cells demonstrate a diminished repopulating capacity relative to that of purified bone marrow-derived hematopoietic stem cells (BM HSCs). It has therefore been unclear whether organ-derived HSCs possess functional properties distinct from those of BM HSCs, or simply have not been purified to a comparable extent. Here we report the identification of a rare subset of cells in adult murine liver that possess potent blood-repopulating potential, approaching that of BM HSCs. The cells, isolated on the basis of dye-efflux activity and CD45 expression (termed CD45(+) liver side population [SP] tip cells), exhibit a surface phenotype similar to that of freshly isolated BM HSCs derived from normal adult animals, but are phenotypically distinct in that they do not express the stem-cell marker c-kit. Single-cell transplantation studies indicate that CD45(+) liver SP tip cells can be generated from BM HSCs, suggesting a relationship between stem-cell populations in the liver and bone marrow compartments. Overall, these studies have important implications for understanding extramedullary hematopoiesis, and may be relevant to current strategies aimed at inducing tolerance to transplanted organs.

Failure of bone marrow to reconstitute lung epithelium.

A new paradigm of epithelial tissue reconstitution has been suggested whereby circulating cells derived from bone marrow contribute to a variety of epithelial cell types. With regard to the lung, several recent reports have used immunofluorescence microscopy to demonstrate engraftment of bone marrow-derived cells as type II pneumocytes, the endogenous progenitors of the lung alveolus. We show here that immunofluorescence microscopy, as has been used in previous reports, cannot reliably identify rare engrafted cells in lung tissue sections after transplantation of bone marrow cells or purified hematopoietic stem cells tracked with ubiquitous labels. We have employed a lineage-specific reporter system based on transgenic mice that express the GFP reporter gene only in lung epithelial cells (surfactant protein C-GFP) to assay for engrafted cells by flow cytometry, histology, and molecular methods. Using this approach to evaluate transplant recipients, including those subjected to bleomycin-induced lung injury, we demonstrate that when autofluorescence, dead cells, and contaminating blood cells are excluded from analysis, there is no detectable reconstitution of lung alveolar epithelial cells by unfractionated bone marrow cells or purified hematopoietic stem cells.

Efficiency of transduction of highly purified murine hematopoietic stem cells by lentiviral and oncoretroviral vectors under conditions of minimal in vitro manipulation.

The development of leukemias in several children with severe combined immunodeficiency disease who were transplanted with retroviral vector-transduced bone marrow cells has renewed concerns about the risks associated with the random integration of proviral sequences into chromosomal DNA. One theoretical way to reduce the risks of insertional mutagenesis would be to employ transduction/transplantation protocols that minimize the total number of genetically modified cells and associated proviral integration "events" introduced into recipients. Toward this end, we have developed a transduction protocol that involves the short-term incubation of highly purified murine stem cells with high-titer recombinant lentivirus vectors in the presence of serum-free medium and the cytokines SCF and TPO. Competitive repopulation studies showed that stem cells transduced in this way possessed the same reconstitutive ability as fresh, unmanipulated cells. Animals transplanted with only 200-2000 transduced cells were efficiently reconstituted with the genetically modified cells, and most hematopoietic cells in the recipients expressed the transgene. In contrast, the use of high-titer oncoretroviral vectors in conjunction with the same transduction/transplantation protocol resulted in only low levels of gene marking in vivo. The use of a similar transduction/transplantation strategy in future clinical studies may offer distinct advantages over current protocols.