Search

The success of chimeric antigen receptor (CAR)–mediated immunotherapy in acute lymphoblastic leukemia (ALL) highlights the potential of T-cell therapies with directed cytotoxicity against specific tumor antigens. The efficacy of CAR T-cell therapy depends on the engraftment and persistence of T cells following adoptive transfer. Most protocols for T-cell engineering routinely expand T cells ex vivo for 9 to 14 days. Because the potential for engraftment and persistence is related to the state of T-cell differentiation, we hypothesized that reducing the duration of ex vivo culture would limit differentiation and enhance the efficacy of CAR T-cell therapy. We demonstrated that T cells with a CAR-targeting CD19 (CART19) exhibited less differentiation and enhanced effector function in vitro when harvested from cultures at earlier (day 3 or 5) compared with later (day 9) timepoints. We then compared the therapeutic potential of early versus late harvested CART19 in a murine xenograft model of ALL and showed that the antileukemic activity inversely correlated with ex vivo culture time: day 3 harvested cells showed robust tumor control despite using a 6-fold lower dose of CART19, whereas day 9 cells failed to control leukemia at limited cell doses. We also demonstrated the feasibility of an abbreviated culture in a large-scale current good manufacturing practice–compliant process. Limiting the interval between T-cell isolation and CAR treatment is critical for patients with rapidly progressing disease. Generating CAR T cells in less time also improves potency, which is central to the effectiveness of these therapies. Cancer Immunol Res; 6(9); 1100–9. ©2018 AACR.

Table S1 and Figures S1 -S8

Huate et al supplemental figures.

Purpose:T cells engineered to express a chimeric antigen receptor (CAR) are a promising cancer immunotherapy. Such targeted therapies have shown long-term relapse-free survival in patients with B-cell leukemia and lymphoma. However, cytokine release syndrome (CRS) represents a serious, potentially life-threatening side effect often associated with CAR T-cell therapy. CRS manifests as a rapid (hyper)immune reaction driven by excessive inflammatory cytokine release, including IFNγ and IL6.Experimental Design:Many cytokines implicated in CRS are known to signal through the JAK-STAT pathway. Here we study the effect of blocking JAK pathway signaling on CAR T-cell proliferation, antitumor activity, and cytokine levels in in vitro and in vivo models.Results:We report that itacitinib, a potent, selective JAK1 inhibitor, was able to significantly and dose-dependently reduce levels of multiple cytokines implicated in CRS in several in vitro and in vivo models. Importantly, we also report that at clinically relevant doses that mimic human JAK1 pharmacologic inhibition, itacitinib did not significantly inhibit proliferation or antitumor killing capacity of three different human CAR T-cell constructs (GD2, EGFR, and CD19). Finally, in an in vivo model, antitumor activity of CD19-CAR T cells adoptively transferred into CD19+ tumor-bearing immunodeficient animals was unabated by oral itacitinib treatment.Conclusions:Together, these data suggest that itacitinib has potential as a prophylactic agent for the prevention of CAR T cell–induced CRS, and a phase II clinical trial of itacitinib for prevention of CRS induced by CAR T-cell therapy has been initiated (NCT04071366).

Prior to adoptive transfer, CAR T cells are activated, lentivirally infected with CAR transgenes, and expanded over 9 to 11 days. An unintended consequence of this process is the progressive differentiation of CAR T cells over time in culture. Differentiated T cells engraft poorly, which limits their ability to persist and provide sustained tumor control in hematologic as well as solid tumors. Solid tumors include other barriers to CAR T cell therapies, including immune and metabolic checkpoints that suppress effector function and durability. Sialic acids are ubiquitous surface molecules with known immune checkpoint functions. The enzyme C. perfringens neuraminidase (CpNA) removes sialic acid residues from target cells, with good activity at physiologic conditions. In combination with galactose oxidase (GO), NA has been found to stimulate T cell mitogenesis and cytotoxicity in vitro. Here we determine whether CpNA alone and in combination with GO promotes CAR T cell antitumor efficacy. We show that CpNA restrains CAR T cell differentiation during ex vivo culture, giving rise to progeny with enhanced therapeutic potential. CAR T cells expressing CpNA have superior effector function and cytotoxicity in vitro. In a Nalm-6 xenograft model of leukemia, CAR T cells expressing CpNA show enhanced antitumor efficacy. Arming CAR T cells with CpNA also enhanced tumor control in xenograft models of glioblastoma as well as a syngeneic model of melanoma. Given our findings, we hypothesize that charge repulsion via surface glycans is a regulatory parameter influencing differentiation. As T cells engage target cells within tumors and undergo constitutive activation through their CARs, critical thresholds of negative charge may impede cell-cell interactions underlying synapse formation and cytolysis. Removing the dense pool of negative cell-surface charge with CpNA is an effective approach to limit CAR T cell differentiation and enhance overall persistence and efficacy.

Purpose: T cells engineered to express a chimeric antigen receptor (CAR) are a promising cancer immunotherapy. Such targeted therapies have shown long-term relapse-free survival in patients with B-cell leukemia and lymphoma. However, cytokine release syndrome (CRS) represents a serious, potentially life-threatening side effect often associated with CAR T-cell therapy. CRS manifests as a rapid (hyper)immune reaction driven by excessive inflammatory cytokine release, including IFNγ and IL6. Experimental Design: Many cytokines implicated in CRS are known to signal through the JAK-STAT pathway. Here we study the effect of blocking JAK pathway signaling on CAR T-cell proliferation, antitumor activity, and cytokine levels in in vitro and in vivo models. Results: We report that itacitinib, a potent, selective JAK1 inhibitor, was able to significantly and dose-dependently reduce levels of multiple cytokines implicated in CRS in several in vitro and in vivo models. Importantly, we also report that at clinically relevant doses that mimic human JAK1 pharmacologic inhibition, itacitinib did not significantly inhibit proliferation or antitumor killing capacity of three different human CAR T-cell constructs (GD2, EGFR, and CD19). Finally, in an in vivo model, antitumor activity of CD19-CAR T cells adoptively transferred into CD19+ tumor-bearing immunodeficient animals was unabated by oral itacitinib treatment. Conclusions: Together, these data suggest that itacitinib has potential as a prophylactic agent for the prevention of CAR T cell–induced CRS, and a phase II clinical trial of itacitinib for prevention of CRS induced by CAR T-cell therapy has been initiated (NCT04071366).

Effective chimeric antigen receptor (CAR)-T cell therapy is dependent on optimal cell culture methods conducive to the activation and expansion of T cells ex vivo, as well as infection with CAR. Media formulations used in CAR-T cell manufacturing have not been optimized for gene delivery, cell expansion, and overall potency. Bioactive components and derivatives that support the generation of functionally-competent T cell progeny with long-lasting persistence are largely undefined. Current media formulations rely on fetal bovine serum (FBS) or human serum (HS), which suffer from a lack of consistency or supply issues. We recognize that components of blood cellular fractions that are absent in serum may have therapeutic value. Here we investigate whether a concentrated growth factor extract, purified from human transfusion grade whole blood fractions, and marketed as PhysiologixTM xeno-free (XF) hGFC (Phx), supports CAR-T cell expansion and function. We show that Phx supports T cell proliferation in clinical and research-grade media. We also show that Phx treatment enhances lentiviral-mediated gene expression across a wide range of multiplicity of infections (MOIs). We compared the ability of anti-GD-2 CAR-T cells expanded ex vivo in medium conditioned with either Phx or HS to clear tumor burden in a human xenograft model of neuroblastoma. We show that T cells expanded in Phx have superior engraftment and potency in vivo, as well as CAR-induced cytolytic activity in vitro. Metabolomic profiling revealed several factors unique to Phx that may have relevance for CAR-T cell preclinical discovery, process development, and manufacturing. In particular, we show that carnosine, a biogenic amine modestly enriched in Phx relative to HS, enhances lentiviral gene delivery in activated T cells. By limiting extracellular acidification, carnosine enhances the metabolic fitness of T cells, shifting their metabolic profile from an acidic, stressed state toward an oxidative, energetic state. These findings are very informative regarding potential derivatives to include in medium customized for gene delivery and overall potency for T cell adoptive immunotherapies., Graphical Abstract, Media formulations used in CAR-T cell therapies have not been optimized for gene delivery, cell expansion, and overall potency. Ghassemi et al. report that anti-GD2 CAR-T cells expanded in medium conditioned with a concentrated growth factor extract have superior engraftment and potency in a human xenograft model of neuroblastoma.

The metabolic milieu of solid tumors provides a barrier to chimeric antigen receptor (CAR) T-cell therapies. Excessive lactate or hypoxia suppresses T-cell growth, through mechanisms including NADH buildup and the depletion of oxidized metabolites. NADH is converted into NAD+ by the enzyme Lactobacillus brevis NADH Oxidase (LbNOX), which mimics the oxidative function of the electron transport chain without generating ATP. Here we determine if LbNOX promotes human CAR T-cell metabolic activity and antitumor efficacy. CAR T-cells expressing LbNOX have enhanced oxygen as well as lactate consumption and increased pyruvate production. LbNOX renders CAR T-cells resilient to lactate dehydrogenase inhibition. But in vivo in a model of mesothelioma, CAR T-cell’s expressing LbNOX showed no increased antitumor efficacy over control CAR T-cells. We hypothesize that T cells in hostile environments face dual metabolic stressors of excessive NADH and insufficient ATP production. Accordingly, futile T-cell NADH oxidation by LbNOX is insufficient to promote tumor clearance.

Chimaeric antigen receptor (CAR) T cells can generate durable clinical responses in B-cell haematologic malignancies. The manufacturing of these T cells typically involves their activation, followed by viral transduction and expansion ex vivo for at least 6 days. However, the activation and expansion of CAR T cells leads to their progressive differentiation and the associated loss of anti-leukaemic activity. Here we show that functional CAR T cells can be generated within 24 hours from T cells derived from peripheral blood without the need for T-cell activation or ex vivo expansion, and that the efficiency of viral transduction in this process is substantially influenced by the formulation of the medium and the surface area-to-volume ratio of the culture vessel. In mouse xenograft models of human leukaemias, the rapidly generated non-activated CAR T cells exhibited higher anti-leukaemic in vivo activity per cell than the corresponding activated CAR T cells produced using the standard protocol. The rapid manufacturing of CAR T cells may reduce production costs and broaden their applicability.

Background & Aim The recent success of immunotherapy using chimeric antigen receptor modified T cells (CAR T) in B-cell malignancies highlights the potential of these cytotoxic “drugs” for cancer therapy. CAR T therapies generally relies upon viral transduction of T cell-receptor (TCR) activated T cell followed by ex vivo expansion of patient-derived T cells over days to weeks prior to infusion. In addition to the required time and labor, we previously reported that TCR/CD3 activation and ex vivo expansion leads to progressive differentiation of the CAR T cells with associated loss of CAR T cell potency. Since cell division is not a prerequisite for lentiviral vector-mediated gene delivery, we hypothesized that elimination of the CD3/CD28 activation step would yield a T cell product with high functional potency. Methods, Results & Conclusion Here, we show that functional CD19-specific CAR T cells (CART19) can be generated in as little as 24 hours using lentiviral vectors without the need for prior T cell activation. We further demonstrate that some of the restriction factors that limit the infection of quiescent T cells can be overcome by changes to the medium formulation. Most importantly, we show that non-activated T cells exhibit potent antitumor function with deep and durable control of leukemia achieved in vivo with greater than 1-log fewer CD19-specific CAR T cells compared with a 9-day process currently used by tisagenlecleucel (Fig. 1). These findings confirmed that as few as an estimated 12,000-32,000 non-activated CAR T cells produced within 24 hours from mononuclear cell collection could eradicate leukemia. These results demonstrate the potential for vastly reducing the time, materials and labor required to generate CAR T cells, which could be especially beneficial in patients with rapidly progressive disease and in resource-poor health care environments.

Background & Aim Effective CAR T-cell therapy is dependent on optimal cell culture methods conducive to the activation and expansion of T cells ex vivo as well as infection with CAR. In this regard, the outcome of the ex-vivo process is largely dependent on the properties of the medium. Media formulations used in CAR T-cell manufacturing have not been optimized either for signaling studies, gene delivery or cell expansion. Bioactive components and derivatives that support the generation of functionally-competent T cell progeny with long-lasting persistence are largely undefined. Current media formulations rely on fetal bovine serum (FBS) or human serum (HS) which suffer from a lack of consistency or supply issues. We recognize that components of blood cellular fractions that are absent in serum may have therapeutic value. Here we investigate if a concentrated growth factor extract, purified from human transfusion grade whole blood fractions, and marketed as PhysiologixTM XF hGFC (Phx), supports CAR T-cell expansion and function. Methods, Results & Conclusion We show that Phx supports T cell proliferation in clinical as well as research-grade media. As cytokine stimulation regulates the exo and endocytotic machinery, we hypothesized that factors unique to the growth factors within blood cell fractions would enhance the dynamic process of viral entry. We show that Phx treatment enhances lentiviral-mediated gene expression across a wide range of multiplicity of infections (MOIs). We compared the ability of anti-GD-2 CAR T-cells expanded ex vivo in medium conditioned with either Phx or HS to clear tumor burden in a human xenograft model of neuroblastoma. We show that CAR T-cells expanded in Phx have superior engraftment and potency in vivo. Metabolomic profiling revealed several factors enriched in a blood cell-derived growth factor concentrate that may enhance lentiviral-mediated gene delivery and CAR T-cell differentiation for preclinical discovery, process development, as well as manufacturing. These findings are also informative regarding potential derivatives to include in medium customized for gene delivery, T cell activation, and expansion for adoptive immunotherapies.

The success of chimeric antigen receptor (CAR)–mediated immunotherapy in acute lymphoblastic leukemia (ALL) highlights the potential of T-cell therapies with directed cytotoxicity against specific tumor antigens. The efficacy of CAR T-cell therapy depends on the engraftment and persistence of T cells following adoptive transfer. Most protocols for T-cell engineering routinely expand T cells ex vivo for 9 to 14 days. Because the potential for engraftment and persistence is related to the state of T-cell differentiation, we hypothesized that reducing the duration of ex vivo culture would limit differentiation and enhance the efficacy of CAR T-cell therapy. We demonstrated that T cells with a CAR-targeting CD19 (CART19) exhibited less differentiation and enhanced effector function in vitro when harvested from cultures at earlier (day 3 or 5) compared with later (day 9) timepoints. We then compared the therapeutic potential of early versus late harvested CART19 in a murine xenograft model of ALL and showed that the antileukemic activity inversely correlated with ex vivo culture time: day 3 harvested cells showed robust tumor control despite using a 6-fold lower dose of CART19, whereas day 9 cells failed to control leukemia at limited cell doses. We also demonstrated the feasibility of an abbreviated culture in a large-scale current good manufacturing practice–compliant process. Limiting the interval between T-cell isolation and CAR treatment is critical for patients with rapidly progressing disease. Generating CAR T cells in less time also improves potency, which is central to the effectiveness of these therapies. Cancer Immunol Res; 6(9); 1100–9. ©2018 AACR.

The recent success of immunotherapy using chimeric antigen receptor modified T cells (CAR T) in B-cell malignancies highlights the potential of these cytotoxic "drugs" for cancer therapy. CAR T therapies generally rely upon manufacturing approaches that include prior T cell activation through engagement of the TCR and costimulatory receptors followed by ex vivo expansion of patient-derived T cells over days to weeks. We previously reported that CD3/CD28 stimulation prior to transduction promotes progressive T cell effector differentiation over time in culture with loss of CAR T cell potency (Ghassemi et al. 2018 PMID: 30030295). Since cell division is not a prerequisite for lentiviral vector-mediated gene delivery, we hypothesized that lentiviral transduction of quiescent T cells without prior activation will enhance engraftment and persistence of CART cells that is associated with long-term leukemia control. Here, we show that functional CD19-specific CAR T cells (CART19) can be generated in as little as 24 hours using lentiviral vectors without the need for prior T cell activation. We showed using a non-optimized process that a mean of 6.5% (range 2%-10%) of freshly isolated quiescent T cells can be transduced using an infrared red fluorescent protein (iRFP)-expressing lentiviral vector with slower kinetics of expression compared with activated T cell transduction (peak at 96 hrs vs. 48 hrs for quiescent and activated T cells, respectively). Although substantially less efficient compared to activated T cells, transduction was detected across all T cells subsets with central memory T cells showing the greatest transduction efficiency with a mean of 4-fold greater transduction compared with naïve T cells. Somewhat unexpectedly, CART19 cells generated from quiescent T cells using a CD19-specific CAR vector showed a 3-5 fold greater transgene expression compared with iRFP vectors transduced at similar MOI. However, we show that CAR expression can occur in quiescent T cells even without reverse transcription or integrase function, so called "pseudotransduction". Importantly, we show that this CAR expression produces T cells with cytolytic activity and effector cytokine production in response to antigen that is similar to activated and transduced CAR T cells. Using the well-characterized Nalm6 model of acute lymphoblastic leukemia, we show that CART19 cells generated by transduction of quiescent T cells for 16 hours followed by washing to remove vector exhibit dose-dependent anti-leukemic activity that is durable with injection of as little as 2x105 total T cells. We estimate the latter to contain ~2x104 T cells with integrated lentiviral vector based upon transduction efficiency determined in studies using non-tumor bearing mice. (Fig 1). In summary, our results support the need for further investigation of CAR T cells that are generated using engineering of quiescent T cells. Taking advantage of the ability of lentiviral vectors to transfer genes to quiescent T cells, the highly abbreviated and simplified manufacturing approach described here has the potential to enhance therapeutic potency while also substantially reducing the materials and labor costs associated with current manufacturing approaches that use activated and expanded T cells. In addition, the rapid nature of this manufacturing has the potential to extend the population of patients that may be treated with these therapies by shortening the interval between aphaeresis collection and re-infusion of CAR T cells, which prevents the treatment of some patients with rapidly progressive disease. Disclosures Ghassemi: Novartis: Patents & Royalties. Milone:Novartis: Patents & Royalties, Research Funding.

Targeting of drug carriers to cell adhesion molecules expressed on endothelial cells (ECs) may improve treatment of diseases involving the vascular endothelium. This is the case for carriers targeted to intercellular adhesion molecule 1 (ICAM-1), an endothelial surface protein overexpressed in many pathologies. In order to optimize our design of anti-ICAM carriers, we have explored in this study the influence of two carrier design parameters on specific and efficient endothelial targeting in vitro and in vivo: carrier dose and density of targeting molecules (antibodies -Ab-) on the carrier surface. Using radioisotope tracing we assessed the role of these parameters on the biodistribution of model polymer carriers targeted to ICAM-1 (125I-anti-ICAM carriers) in mice. Increasing the carrier dose enhanced specific accumulation in the lung vasculature (a preferential endothelial target) and decreased non-specific hepatic and splenic uptake. Increasing the Ab density enhanced lung accumulation with minimally reduced liver and spleen uptake. These studies account for the influence of blood hydrodynamic forces on carrier binding to endothelium, relevant to arterioles, venules and larger vessels. Yet, carriers may rather bind to the extensive capillary bed where shear stress is minimal. We used fluorescence microscopy to determine binding kinetics of FITC-labeled anti-ICAM carriers in static conditions, at the threshold found in vivo and conditions mimicking low vs high ICAM-1 expression on quiescent vs activated ECs. Binding to activated ECs reached similar saturation with all tested Ab densities and carrier concentrations. In quiescent cells, carriers reached ~3-fold lower binding saturation, even at high carrier concentration and Ab density, and carriers with low Ab density did not reach saturation, reflecting avidity below threshold. Binding kinetics was positively regulated by anti-ICAM carrier concentration and Ab density. Counterintuitively, binding was faster in quiescent ECs (except for carriers with high Ab density and concentration), likely due to fast saturation of fewer binding sites on these cells. These results will guide optimization of ICAM-1-targeted carriers, e.g., in the context of targeting healthy vs diseased endothelium for prophylactic vs therapeutic interventions.

Animals capable of regenerating multiple tissue types, organs, and appendages after injury are common yet sporadic and include some sponge, hydra, planarian, and salamander (i.e., newt and axolotl) species, but notably such regenerative capacity is rare in mammals. The adult MRL mouse strain is a rare exception to the rule that mammals do not regenerate appendage tissue. Certain commonalities, such as blastema formation and basement membrane breakdown at the wound site, suggest that MRL mice may share other features with classical regenerators. As reported here, MRL fibroblast-like cells have a distinct cell-cycle (G2/M accumulation) phenotype and a heightened basal and wound site DNA damage/repair response that is also common to classical regenerators and mammalian embryonic stem cells. Additionally, a neutral and alkaline comet assay displayed a persistent level of intrinsic DNA damage in cells derived from the MRL mouse. Similar to mouse ES cells, the p53-target p21 was not expressed in MRL ear fibroblasts. Because the p53/p21 axis plays a central role in the DNA damage response and cell cycle control, we directly tested the hypothesis that p21 down-regulation could functionally induce a regenerative response in an appendage of an otherwise nonregenerating mouse strain. Using the ear hole closure phenotype, a genetically mapped and reliable quantitative indicator of regeneration in the MRL mouse, we show that the unrelated Cdkn1a tmi/Tyj /J p21 −/− mouse (unlike the B6129SF2/J WT control) closes ear holes similar to MRL mice, providing a firm link between cell cycle checkpoint control and tissue regeneration.

Whereas amphibians regenerate lost appendages spontaneously, mammals generally form scars over the injury site through the process of wound repair. The MRL mouse strain is an exception among mammals because it shows a spontaneous regenerative healing trait and so can be used to investigate proregenerative interventions in mammals. We report that hypoxia-inducible factor 1α (HIF-1α) is a central molecule in the process of regeneration in adult MRL mice. The degradation of HIF-1α protein, which occurs under normoxic conditions, is mediated by prolyl hydroxylases (PHDs). We used the drug 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA), a PHD inhibitor, to stabilize constitutive expression of HIF-1α protein. A locally injectable hydrogel containing 1,4-DPCA was designed to achieve controlled delivery of the drug over 4 to 10 days. Subcutaneous injection of the 1,4-DPCA/hydrogel into Swiss Webster mice that do not show a regenerative phenotype increased stable expression of HIF-1α protein over 5 days, providing a functional measure of drug release in vivo. Multiple peripheral subcutaneous injections of the 1,4-DPCA/hydrogel over a 10-day period led to regenerative wound healing in Swiss Webster mice after ear hole punch injury. Increased expression of the HIF-1α protein may provide a starting point for future studies on regeneration in mammals.

We previously demonstrated that after a severe cryoinjury to the right ventricle of the heart, adult MRL mice display structural and functional recovery with myocardial tissue replacement resembling that seen in amphibians. The control non-regenerating adult C57BL/6 (B6) mouse shows a predominant scar response. In the present study, radiation chimeras reconstituted with fetal liver cells from either healer MRL or nonhealer B6 mice were generated to test for a transfer of phenotype. Allogeneic MRL fetal liver cells were injected into x-irradiated (9 Gy) B6 mice and B6 fetal liver cells were injected into x-irradiated MRL mice. In these allogeneic chimeras, the healing response to cardiac cryoinjury was predominantly of the donor phenotype. Thus, MRL fetal liver cells transferred the healing phenotype to the B6 nonhealer with the appearance of Y-chromosome positive, donor-derived cardiomyocytes in the injury site and MRL-like healing with little scar. Similarly, B6 fetal liver cells transferred the nonhealing phenotype to the MRL with little cardiomyocyte growth and an acellular B6-like scar. These results are in contrast to the ear hole closure response which was of the recipient phenotype. We conclude that, in the case of the heart, fetal liver-derived stem cells regulate regenerative healing.

The ability to regenerate tissues and limbs in its most robust form is seen in many non–mammalian species. The serendipitous discovery that the MRL mouse has a profound capacity for regeneration in some ways rivalling the classic newt and axolotl species raises the possibility that humans, too, may have an innate regenerative ability. The adult MRL mouse regrows cartilage, skin, hair follicles and myocardium with near perfect fidelity and without scarring. This is seen in the ability to close through–and–through ear holes, which are generally used for lifelong identification of mice, and the anatomic and functional recovery of myocardium after a severe cryo–injury. We present histological, biochemical and genetic data indicating that the enhanced breakdown of scar–like tissue may be an underlying factor in the MRL regenerative response. Studies as to the source of the cells in the regenerating MRL tissue are discussed. Such studies appear to support multiple mechanisms for cell replacement.

Over the past several years many mechanisms by which myocardial replacement could be achieved have been described. These include resident cardiac stem cells or circulating stem cells that can either differentiate into, or fuse to cardiomyocytes, or mature cells that can transdifferentiate into cardiomyocytes. However, the fact remains that after injury to the heart, the overriding response is scar formation with little myocardial replacement. One exception to this response is the MRL mouse, which heals with little scarring and shows nearly full myocardial replacement after injury. Results obtained with this model will be discussed.

The reaction of myocardium to injury is generally characterized by a tissue repair mechanism that is initially beneficial in maintaining tissue integrity but which ultimately has a debilitating effect on cardiac function. The components of this type of wound healing response in the mammalian heart have been the subject of extensive study. Far less understood, however, are the dynamics of inflammation, ECM remodeling and scar formation at the site of injury, as well as compensatory adaptations of adjacent uninjured myocardium such as angiogenesis, cadiomyocyte hypertrophy, and apoptosis in the regenerative response to a myocardial injury. Recent observations in the MRL strain of mice indicate a capacity to heal myocardial injury with a rapid resolution of granulation tissue and little scar tissue, an increase of cardiomyocyte BrdU incorporation and restoration of myocardial architecture/function. The role of stem cells in this process remains to be elucidated for MRL mice. This response to injury, however, suggests epimorphic regeneration rather than repair.

Activated T lymphocytes are present in human atherosclerotic lesions and autoantibodies to antigens within lesions have been detected in serum, but the roles of the cellular and humoral immune systems in atherogenesis have not been determined. The effect of total lymphocyte deficiency on atherogenesis was investigated by crossing apo E-deficient mice (which develop atherosclerosis resembling human disease) with mice deficient in RAG2 (which is required for normal B and T lymphocyte development). Mice were placed on a fat- and cholesterol-enriched diet for 12 wk. RAG2-deficient mice had no serum autoantibodies, in contrast to the high titers in RAG2+/- littermates. There were no T lymphocytes and a markedly reduced number of MHC class II-positive macrophages in atherosclerotic lesions of RAG2-deficient mice. Despite these differences, RAG2-deficient mice developed atherosclerosis similar in extent to that in immunocompetent littermates, based on quantification by two independent methods. In conclusion, the absence of autoantibodies and T lymphocytes did not influence the extent of aortic atherosclerotic lesions in apo E-deficient mice.

Mouse strains C57BL/6 (B6) and MRL were studied by whole mouse genome chip microarray analyses of RNA isolated from amputation sites at different times pre- and postamputation at the midsecond phalange of the middle digit. Many keratin genes were highly differentially expressed. All keratin genes were placed into three temporal response classes determined by injury/preinjury ratios. One class, containing only Krt6 and Krt16, were uniquely expressed relative to the other two classes and exhibited different temporal responses in MRL vs. B6. Immunohistochemical staining for Krt6 and Krt16 in tissue sections, including normal digit, flank skin, and small intestine, and from normal and injured ear pinna tissue exhibited staining differences in B6 (low) and MRL (high) that were consistent with the microarray results. Krt10 staining showed no injury-induced differences, consistent with microarray expression. We analyzed Krt6 and Krt16 gene association networks and observed in uninjured tissue several genes with higher expression levels in MRL, but not B6, that were associated with the keratinocyte activated state: Krt6, Krt16, S100a8, S100a9, and Il1b; these data suggest that keratinocytes in the MRL strain, but not in B6, are in an activated state prior to wounding. These expression levels decreased in MRL at all times postwounding but rose in the B6, peaking at day 3. Other keratins significantly expressed in the normal basal keratinocyte state showed no significant strain differences. These data suggest that normal MRL skin is in a keratinocyte activated state, which may provide it with superior responses to wounding.

Myotonic dystrophy (DM) is an autosomal dominant disorder resulting from the expansion of a CTG repeat in the 3' untranslated region of a putative protein kinase (DMPK). To elucidate the role of DMPK in DM pathogenesis we have developed Dmpk deficient (Dmpk-/-) mice. Dmpk-/-mice develop a late-onset, progressive skeletal myopathy that shares some pathological features with DM. Muscles from mature mice show variation in fibre size, increased fibre degeneration and fibrosis. Adult Dmpk-/-mice show ultrastructural changes in muscle and a 50% decrease in force generation compared to young mice. Our results indicate that DMPK may be necessary for the maintenance of skeletal muscle structure and function and suggest that a decrease in DMPK levels may contribute to DM pathology.

The expression of a ski transgene in the bind leg muscles of mice follows a spatial and temporal pattern reminiscent of the pattern of myogenic development. Anterior muscles, which are formed earliest during development, are also the first muscles to express ski mRNA. Muscles derived from the posterior muscle group, formed later during development, exhibit delayed expression of ski mRNA. In addition, there is regional variation in ski mRNA levels within a particular muscle. Superficial regions of fast muscles, which contain a large percentage of type IIb fibers and have a high ATPase activity, express a higher level of ski mRNA than the deep portions of the same muscles. The deep regions contain a lower percentage of type IIb fibers and lower ATPase activity. The soleus, a slow muscle composed predominantly of type I fibers, expresses low ATPase activity and contains much lower levels of ski mRNA. mRNA from the ski transgene is also expressed at high levels in the osteocytes of the leg bones of 15-day and older transgenic mice. High levels of Ski protein is present in the osteocytes of the leg bones. ski expression appears to cause remodeling of the tibia and fibula. The cross-sectional area of the tibia and fibula of ski transgenic mice is significantly decreased compared to controls. X-rays of the skeletons of ski transgenic mice suggest that the bones of the entire skeleton are thinner than the bones in normal mice. Pathological stress fractures were found in several bones in the ski transgenic mice.

Digit re-growth following amputation injury proximal to the first phalangeal joint is not a property of mammalian wound healing. However, the regenerative potential observed in the MRL mouse invites a re-examination of this rule. In this study, healing was assessed in three mouse strains after amputation midway through the second phalangeal bone. Three distinct outcomes were observed though evidence for re-growth was observed only in the MRL mouse. Here, a blastema-like structure was seen along with apparent chondrogenesis, consistent with a histological profile of a regenerative response to injury. Analysis of trichrome staining and basement membrane changes, proliferation and apoptosis indicated that these processes contributed to the formation of new digit tissue. On the other hand, SW and B6 digits did not show evidence of growth with little mesenchymal BrdU incorporation or phosphorylation of H3.

ALTHOUGH murine X-linked muscular dystrophy (mdx) and Duchenne muscular dystrophy (DMD) are genetically homologous and both characterized by a complete absence of dystrophin1,2, the limb muscles of adult mdx mice suffer neither the detectable weakness nor the progressive degeneration that are features of DMD3–8. Here we show that the mdx mouse diaphragm exhibits a pattern of degeneration, fibrosis and severe functional deficit comparable to that of DMD limb muscle, although adult mice show no overt respiratory impairment. Progressive functional changes include reductions in strength (to 13.5% of control by two years of age), elasticity, twitch speed and fibre length. The collagen density rises to at least seven times that of control diaphragm and ten times that of mdx hind-limb muscle. By 1.5 years of age, similar but less severe histological changes emerge in the accessory muscles of respiration. On the basis of these findings, we propose that dystrophin deficiency alters the threshold for work-induced injury. Our data provide a quantitative framework for studying the pathogenesis of dystrophy and extend the application of the mdx mouse as an animal model.

Endocytosis in endothelial cells (ECs) is important for many biomedical applications, including drug delivery by nano- and microscale carriers. However, little is known about how carrier geometry influences endothelial drug targeting, intracellular trafficking, and effects. We studied this using prototype polymer carriers of various sizes (0.1-10 mum) and shapes (spheres versus elliptical disks). Carriers were targeted to intercellular adhesion molecule 1 (ICAM-1), a transmembrane glycoprotein that is upregulated in many pathologies and used as a target for intraendothelial drug delivery. ECs internalized anti-ICAM-coated carriers of up to several microns in size via cell adhesion molecule-mediated endocytosis. This pathway is distinct from caveolar and clathrin endocytosis that operate for submicron-size objects. Carrier geometry was found to influence endothelial targeting in the vasculature, and the rate of endocytosis and lysosomal transport within ECs. Disks had longer half-lives in circulation and higher targeting specificity in mice, whereas spheres were endocytosed more rapidly. Micron-size carriers had prolonged residency in prelysosomal compartments, beneficial for endothelial antioxidant protection by delivered catalase. Submicron carriers trafficked to lysosomes more readily, optimizing effects of acid sphingomyelinase (ASM) enzyme replacement in a model of lysosomal storage disease. Therefore, rational design of carrier geometry will help optimize endothelium-targeted therapeutics.

Type B Niemann-Pick disease (NPD) is a multiorgan system disorder caused by a genetic deficiency of acid sphingomyelinase (ASM), for which lung is an important and challenging therapeutic target. In this study, we designed and evaluated new delivery vehicles for enzyme replacement therapy of type B NPD, consisting of polystyrene and poly(lactic-coglycolic) acid polymer nanocarriers targeted to intercellular adhesion molecule (ICAM)-1, an endothelial surface protein up-regulated in many pathologies, including type B NPD. Real-time vascular imaging using intravital microscopy and postmortem imaging of mouse organs showed rapid, uniform, and efficient binding of fluorescently labeled ICAM-1-targeted ASM nanocarriers (anti-ICAM/ASM nanocarriers) to endothelium after i.v. injection in mice. Fluorescence microscopy of lung alveoli actin, tissue histology, and 125I-albumin blood-to-lung transport showed that anti-ICAM nanocarriers cause neither detectable lung injury, nor abnormal vascular permeability in animals. Radioisotope tracing showed rapid disappearance from the circulation and enhanced accumulation of anti-ICAM/125I-ASM nanocarriers over the nontargeted naked enzyme in kidney, heart, liver, spleen, and primarily lung, both in wild-type and ASM knockout mice. These data demonstrate that ICAM-1-targeted nanocarriers may enhance enzyme replacement therapy for type B NPD and perhaps other lysosomal storage disorders.

Coupling tissue-type plasminogen activator (tPA) to carrier red blood cells (RBC) prolongs its intravascular life span and permits its use for thromboprophylaxis. Here, we studied the susceptibility of RBC/tPA to PA inhibitors including plasminogen activator inhibitor-1 (PAI-1) that constrain its activity and may reduce the duration of its effect. Despite lesser spatial and diffusional limitations, soluble tPA was far less effective than RBC/tPA in dissolving clots formed in vitro from blood of wild-type (WT) mice (40 versus 80% lysis at equal doses of tPA). Furthermore, after i.v. injection, soluble tPA lost activity faster in transgenic mice expressing a high level of PAI-1 than in WT mice, whereas the activity of RBC/tPA was unaffected. PAI-1 inactivated soluble tPA at equimolar ratios in vitro, but it had no effect on the amidolytic or fibrinolytic activity of RBC/tPA. RBC/tPA was also more resistant than soluble tPA to in vitro inhibition by other serpins (alpha2-macroglobulin and alpha1-antitrypsin) and pathologically high levels of glucose. However, coupling to RBC did not protect a truncated tPA mutant, Retavase, from plasma inhibitors. Chemical removal of the RBC glycocalyx negated tPA protection from inhibitors: tPA coupled to glycocalyx-stripped RBC bound twice as much 125I-PAI-1 as did tPA coupled to naive RBC, and susceptibility of the bound tPA to inhibition by PAI-1 was restored. Thus, the RBC glycocalyx protects RBC-coupled tPA against inhibition. Resistance to high levels of inhibitors in vivo contributes to the potential utility of RBC/tPA for thromboprophylaxis.

A particular mouse strain, the MRL mouse, has been shown to have unique healing properties that show normal replacement of tissue without scarring. The serendipitous discovery that the MRL mouse has a profound capacity for regeneration in some ways rivaling the classic newt and axolotl species raises the possibility that humans, too, may have an innate regenerative ability. We propose this mouse as a model for continuous regeneration with possible life-extending properties. We will use the classical "immortal" organism, the hydra, for comparison and examine those key phenotypes that contribute to their immortality as they are expressed in the MRL mouse versus control mouse strains. The phenotypes to be examined include the rate of proliferation and the rate of cell death, which leads to a continual turnover in cells without an increase in mass.

Targeting of diagnostic and therapeutic agents to endothelial cells (ECs) provides an avenue to improve treatment of many maladies. For example, intercellular adhesion molecule 1 (ICAM-1), a constitutive endothelial cell adhesion molecule up-regulated in many diseases, is a good determinant for endothelial targeting of therapeutic enzymes and polymer nanocarriers (PNCs) conjugated with anti-ICAM (anti-ICAM/PNCs). However, intrinsic and extrinsic factors that control targeting of anti-ICAM/PNCs to ECs (e.g., anti-ICAM affinity and PNC valency and flow) have not been defined. In this study we tested in vitro and in vivo parameters of targeting to ECs of anti-ICAM/PNCs consisting of either prototype polystyrene or biodegradable poly(lactic-coglycolic) acid polymers (approximately 200 nm diameter spheres carrying approximately 200 anti-ICAM molecules). Anti-ICAM/PNCs, but not control IgG/PNCs 1) rapidly (t1/2 approximately 5 min) and specifically bound to tumor necrosis factor-activated ECs in a dose-dependent manner (Bmax approximately 350 PNC/cell) at both static and physiological shear stress conditions and 2) bound to ECs and accumulated in the pulmonary vasculature after i.v. injection in mice. Anti-ICAM/PNCs displayed markedly higher EC affinity versus naked anti-ICAM (Kd approximately 80 pM versus approximately 8 nM) in cell culture and, probably because of this factor, higher value (185.3 +/- 24.2 versus 50.5 +/- 1.5% injected dose/g) and selectivity (lung/blood ratio 81.0 +/- 10.9 versus 2.1 +/- 0.02, in part due to faster blood clearance) of pulmonary targeting. These results 1) show that reformatting monomolecular anti-ICAM into high-affinity multivalent PNCs boosts their vascular immuno-targeting, which withstands physiological hydrodynamics and 2) support potential anti-ICAM/PNCs utility for medical applications.

The ability to regenerate is thought to be a lost phenotype in mammals, though there are certainly sporadic examples of mammalian regeneration. Our laboratory has identified a strain of mouse, the MRL mouse, which has a unique capacity to heal complex tissue in an epimorphic fashion, i.e., to restore a damaged limb or organ to its normal structure and function. Initial studies using through-and-through ear punches showed rapid full closure of the ear holes with cartilage growth, new hair follicles, and normal tissue architecture reminiscent of regeneration seen in amphibians as opposed to the scarring usually seen in mammals. Since the ear hole closure phenotype is a quantitative trait, this has been used to show—through extensive breeding and backcrossing—that the trait is heritable. Such analysis reveals that there is a complex genetic basis for this trait with multiple loci. One of the major phenotypes of the MRL mouse is a potent remodeling response with the absence or a reduced level of scarring. MRL healing is associated with the upregulation of the metalloproteinases MMP-2 and MMP-9 and the downregulation of their inhibitors TIMP-2 and TIMP-3, both present in inflammatory cells such as neutrophils and macrophages. This model has more recently been extended to the heart. In this case, a cryoinjury to the right ventricle leads to near complete scarless healing in the MRL mouse whereas scarring is seen in the control mouse. In the MRL heart, bromodeoxyuridine uptake by cardiomyocytes filling the wound site can be seen 60 days after injury. This does not occur in the control mouse. Function in the MRL heart, as measured by echocardiography, returns to normal.

The reaction of cardiac tissue to acute injury involves interacting cascades of cellular and molecular responses that encompass inflammation, hormonal signaling, extracellular matrix remodeling, and compensatory adaptation of myocytes. Myocardial regeneration is observed in amphibians, whereas scar formation characterizes cardiac ventricular wound healing in a variety of mammalian injury models. We have previously shown that the MRL mouse strain has an extraordinary capacity to heal surgical wounds, a complex trait that maps to at least seven genetic loci. Here, we extend these studies to cardiac wounds and demonstrate that a severe transmural, cryogenically induced infarction of the right ventricle heals extensively within 60 days, with the restoration of normal myocardium and function. Scarring is markedly reduced in MRL mice compared with C57BL/6 mice, consistent with both the reduced hydroxyproline levels seen after injury and an elevated cardiomyocyte mitotic index of 10–20% for the MRL compared with 1–3% for the C57BL/6. The myocardial response to injury observed in these mice resembles the regenerative process seen in amphibians.

The chicken c-ski gene expresses at least three alternatively spliced messages. Transgenic mice expressing proteins from cDNA corresponding to two of these messages (FB27 and FB29) under the control of a murine sarcoma virus (MSV) long terminal repeat (LTR) express the transgene in skeletal muscle and develop a muscular phenotype. Both a biologically active form of c-ski and the MSV LTR are required for the development of the muscular phenotype. The normal c-ski gene linked to two other tissue-specific promoters failed to induce muscle growth in transgenic mice, as did an inactive mutant of c-ski expressed under the control of the MSV LTR.

Limb-girdle muscular dystrophies 2C-F represent a family of autosomal recessive diseases caused by defects in sarcoglycan genes. The cardiomyopathic hamster is a naturally occurring model for limb-girdle muscular dystrophy caused by a primary deficiency in delta-sarcoglycan. We show here that acute sarcolemmal disruption occurs in this animal model during forceful muscle contraction. A recombinant adeno-associated virus vector encoding human delta-sarcoglycan conferred efficient and stable genetic reconstitution in the adult cardiomyopathic hamster when injected directly into muscle. A quantitative assay demonstrated that vector-transduced muscle fibers are stably protected from sarcolemmal disruption; there was no associated inflammation or immunologic response to the vector-encoded protein. Efficient gene transduction with rescue of the sarcoglycan complex in muscle fibers of the distal hindlimb was also obtained after infusion of recombinant adeno-associated virus into the femoral artery in conjunction with histamine-induced endothelial permeabilization. This study provides a strong rationale for the development of gene therapy for limb-girdle muscular dystrophy.

In patients with severe chronic obstructive pulmonary disease, the diaphragm undergoes physiologic adaptations characterized by an increase in energy expenditure and relative resistance to fatigue. We hypothesized that these physiologic characteristics would be associated with structural adaptations consisting of an increased proportion of less-fatigable slow-twitch muscle fibers and slow isoforms of myofibrillar proteins.We obtained biopsy specimens of the diaphragm from 6 patients with severe chronic obstructive pulmonary disease (mean [+/-SE] forced expiratory volume in one second, 33+/-4 percent of the predicted value; residual volume, 259+/-25 percent of the predicted value) and 10 control subjects. The proportions of the various isoforms of myosin heavy chains, myosin light chains, troponin, and tropomyosin were determined by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. We also used immunocytochemical techniques to determine the proportions of the various types of muscle fibers.The diaphragm-biopsy specimens from the patients had higher percentages of slow myosin heavy chain I (64+/-3 vs. 45+/-2 percent, P0.001), and lower percentages of fast myosin heavy chains IIa (29+/-3 vs. 39+/-2 percent, P=0.01) and IIb (8+/-1 vs. 17+/-1 percent, P0.001) than the diaphragms of the controls. Similar differences were noted when immunohistochemical techniques were used to compare the percentages of these fiber types in the two groups. In addition, the patients had higher percentages of the slow isoforms of myosin light chains, troponins, and tropomyosin, whereas the controls had higher percentages of the fast isoforms of these proteins.Severe chronic obstructive pulmonary disease increases the slow-twitch characteristics of the muscle fibers in the diaphragm, an adaptation that increases resistance to fatigue.

The control of c-ski transgene expression and muscle hypertrophy have been investigated in transgenic mice. In adult animals, the level of transgene expression is linked to the specialized phenotype of individual muscles, high levels occur in fast muscles and significantly lower levels in muscles with high metabolic activity (diaphragm, soleus). These findings have led us to propose that a threshold must be passed before ski-induced growth can occur. We now show that within fast muscles, induced hypertrophy uniquely involves IIb fibers. This pattern of expression is under development control; levels of c-ski mRNA are low in all muscles at birth. In the diaphragm, there is a sevenfold increase in c-ski message levels between 5 d and maturity. By contrast, in fast extensor digitorum longus and anterior tibial muscles, there is a 24-fold increase in levels between 5 and 12 d postpartum. Muscle hypertrophy and antibody staining for c-ski protein in myofiber nuclei emerge concurrently. This pattern of c-ski expression parallels the appearance of IIb myosin heavy chain transcripts (Wydert et al., 1987) and differentiation of IIb fibers, suggesting that amplification of c-ski mRNA levels is linked to the development of IIb fiber specialization. Manipulations that are known to perturb IIb fiber development, neonatal denervation, and neonatally induced hypothyroidism inhibit high levels of c-ski expression and hypertrophy. In the adult fast EDL, denervation leads to rapid atrophy of IIb fibers and a significant decline in levels of c-ski mRNA. The results suggest that the environment of differentiated IIb fibers permits the expression of high levels of c-ski mRNA and this, in turn, induces hypertrophy.

Using a variety of techniques, we have demonstrated the presence of at least two fibre types in Limulus median telson levator muscle. By light and electron microscopy, large (2,156 microns 2 mean cross-sectional area) fibres have A-bands of 4.1 microns, one-half I bands of 2.15 microns and Z lines less than or equal to 0.5 microns in width. Few mitochondria are found in these fibres, which comprise 54% of those present in a given microscope field and which occupy 82% of the total cross-sectional area. Small fibres (484 microns 2 mean cross-sectional area) have A bands of 6.3 microns, one-half I bands of 3.1 microns and Z lines between 0.5 and 1.0 microns in width and are rich in mitochondria. Although small fibres comprise nearly one-half (46%) of the fibres in a field, they occupy only 18% of the total cross-sectional area. Histochemical staining for alkaline-stable myofibrillar ATPase activity and mitochondrial reduced beta-nicotinamide adenine nucleotide (beta-NADH) tetrazolium reductase activity confirms the presence of two fibre types. The large fibres react positively for the myofibrillar ATPase activity and negatively for the mitochondrial enzyme activity. The reverse is seen with the small fibres. Some fibres of intermediate size, having intermediate staining characteristics, were also observed. Native gel electrophoresis of both myofibrillar and purified myosin preparations supports the observed differences in myofibrillar ATPase activity in that two myosin isozymes are resolved on pyrophosphate gels. Although the thick filaments isolated from unstimulated small fibres are longer (greater than 6.0 microns) than those isolated from unstimulated large fibres (4.26 microns), all have a similar appearance with respect to the arrangement of myosin heads on their surfaces, and similar diameters. The implications of the observed heterogeneity of fibre types is discussed with reference to previously reported phenomena in Limulus telson muscle, including changes in length of thick filaments on fibre stimulation and the shape of the length-tension curve obtained from fibre bundles.