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M. Hetzel et al.
analysis of pulmonary engraftment and the effect on the various disease parameters. Moreover, in the current study, pronounced therapeutic efficacy was shown for prolonged periods employing the 9-month time point or secondary HSCT as read-outs. Thus, our data demon- strate the profound potential of HSC-based therapies for the treatment of herPAP.
These findings have considerable relevance for the future treatment of herPAP patients. So far, therapy of herPAP patients is symptomatic only, and combines vigor- ous antibiotic prophylaxis and treatment of pulmonary infections with repetitive whole lung lavage, a highly invasive procedure associated with considerable cardio- vascular risks.1,3,5 Importantly, alloHSCT or HSC-GT, which meanwhile represent fairly established therapeutic concepts for other congenital diseases affecting the lym- phohematopoietic system, were considered problematic in herPAP patients due to the severe lung phenotype that complicates the preconditioning procedure.3 On the other hand, reduced-intensity conditioning regimens, improve- ments in diagnosis, and treatment of bacterial, fungal and viral infections, as well as better HLA-matching, have con- siderably advanced the field of HSCT, and successful alloHSCT has recently been described in two patients suf- fering from secondary PAP due to primary immunodefi- ciencies,10 as well as in a herPAP patient.11 Moreover, new therapeutic strategies for herPAP targeting cholesterol homeostasis19,22 may ameliorate symptoms in PAP patients, and, at least in murine disease models, direct intrapulmonary transplantation of macrophages has been shown to improve substantially (if not cure) herPAP dis- ease.12-16 Given this, HSCT-based treatment approaches increasingly appear to be a realistic scenario, and in this context, HSC-GT avoids the critical side effects of alloHSCT such as graft-versus-host disease or graft rejec- tion, and might also allow for less toxic preconditioning regimens.
In the current study, we have utilized Csf2rb-deficient mice primarily based on availability. However, given the almost identical clinical presentation of CSF2RA- and CSF2RB-deficient herPAP patients,23 the HSC-GT based treatment approach presented here should apply to both molecular types of herPAP. Of note, recently also Csf2ra- deficient mice have been generated, and these are now available for detailed efficacy and safety testing also for Csf2ra expression vectors.24,25
In our study, we employed a 3rd generation, safety- improved SIN vector design, which is also used in current HSC-GT-based approaches targeting other monogenic diseases affecting the lymphohematopoietic system, such as X-linked- or adenosine deaminase-deficient severe combined immunodeficiency (X-SCID, ADA-SCID),26,27 Wiskott Aldrich syndrome (WAS),28 metachromatic leukodystrophy,29 or hemoglobinopathies such as β-tha- lassemia and sickle cell disease.30,31 Importantly, in the past, a number of phase I/II clinical studies using HSC-GT approaches and LTR-driven transgene expression suffered from insertional mutagenesis and leukemia induced by transactivation of oncogenes via retroviral enhancer sequences.32-34 In contrast, modern SIN alpha-, or gamma- retroviral, as well as lentiviral vector constructs, harbor inactivating mutations in their LTR and employ internal promoters usually derived from house-keeping genes for transgene expression. Meanwhile, more than 100 patients have undergone HSC-GT employing safety modified SIN
vectors, such as the one used in our study, without a single event of insertional leukemia. This covers observation periods extending up to ten years in individual patients.
In our model, low VCN in BM cells ranging from 0.02 to 0.86 were sufficient to provide pronounced clinical bene- fit, which represents an additional safety feature as low VCN reduce the risk of insertion-triggered mutagenic events. Moreover, it corroborates data from GM-CSFR- deficient cells where single copy CSF2RA expression from an EFS-driven lentiviral construct was sufficient to initiate GM-CSFR downstream signaling.35 Interestingly, corrected cells with VCN of up to 7.32 were recruited to and enriched in the lungs, where they exerted their therapeu- tic effects. This observation is most likely due to the increased pulmonary cytokine levels in herPAP and the feedback mechanism by which cytokines, and in particu- lar GM-CSF, regulate the AM pool.23
Recovery of the AM pool following injection of HSC occurred rapidly and resulted in an intra-alveolar cell pop- ulation displaying the AM typical Siglec-F+CD11c+ pheno- type 4-8 weeks after transplantation recapitulating the data from other murine HSC transplant models.36,37 This time course also mimics quite well the clinical situation following alloHSCT, describing the emergence of donor type AM after 6-8 weeks.11,38 Development of AM from Siglec-F–CD11c– peripheral blood monocytes to mature Siglec-F+CD11c+ AM, as observed in our model, closely recapitulates the physiological development of AM from fetal monocytes in the early postnatal period. During fetal development, an intermediate pre-AM stage seems to be employed, which is no longer detectable once AM differ- entiation is completed.39 Moreover, similar to early post- natal AM development, cytokines such as M-CSF, and par- ticularly GM-CSF, play crucial roles in the generation and maintenance of the AM pool.23,39,40 In this respect, the pro- found reduction of bronchoalveolar M-CSF as well as GM-CSF levels observed in our studies reflect the (auto)- regulatory reaction to the successful pulmonary engraft- ment and differentiation of AM. It remains unknown whether the rapid recovery of the AM compartment in our studies is due to progenitors generated in the murine BM following HSC engraftment or is achieved directly from primitive hematopoietic cells present in the graft.
Thus, in summary, we provide evidence that 3rd genera- tion SIN lentiviral vector-based HSC-GT effectively reverses the GM-CSF-dependent disease pathology and normalizes, or at least significantly improves, critical dis- ease-related parameters in a relevant murine model of herPAP. On these grounds, we would advocate HSC-GT as an effective and cause-directed therapeutic approach for herPAP patients, which in the clinical setting should offer the chance of long-lasting improvement of symp- toms, if not a definitive cure for herPAP patients.
Acknowledgments
The authors would like to thank M. Ballmaier (Cell Sorting Facility, Hannover Medical School) for scientific support and D. Lüttge, E. Janosz, J. Mischke (Institute of Experimental Hematology, Hannover Medical School), P. Felsch, S. Eilert, and A. Kanwischer (Department of Nuclear Medicine, Hannover Medical School) for experimental and technical support. Funding
This work was supported by grants from the Else Kröner- Fresenius-Stiftung (2013_A24 to T.M. and 2015_A92 to NL),
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