Blood -- Restoration of NET formation by gene therapy in CGD controls aspergillosis

Blood, Vol. 114, Issue 13, 2619-2622, September 24, 2009

Restoration of NET formation by gene therapy in CGD controls aspergillosis
Blood Bianchi et al. 114: 2619

Supplemental materials for: Bianchi et al

Patient description and gene therapy
A 3-year-old boy suffered from a severe oxygen-dependent bilateral Aspergillus nidulans lung infection, requiring partial resection of the left lower lung lobe. He was diagnosed with X-linked CGD (CYBB EX1_3del) with complete absence of gp91^phox protein expression and received prophylaxis with itraconazole and Co-trimoxazole. The infection was non-responsive to treatment with IV voriconazole, caspofungine, and granulocyte transfusions, requiring bone marrow transplantation. There was no human leukocyte antigen (HLA)-identical sibling or unrelated donor, and his poor clinical condition excluded transplantation from an HLA-A disparate donor.

The boy was treated with gene therapy at age 8⁷/₁₂ years with a monocistronic LTR-driven gammaretroviral SF71gp91^phox vector¹ under a protocol approved by the ethics review board of the University Children's Hospital Zurich and the Swiss Expert Committee for Bio-Safety after written informed consent of the parents. Collection of CD34⁺ cells, transduction, conditioning with low-dose liposomal busulfan IV (8.8 mg/KG), and clinical follow up were performed as described.¹ Treatment with voriconazole was continued throughout and has not yet been tapered.

To increase engraftment rate,^2,3 CD34⁺ cells were transduced twice. The first batch (A) was delivered intravenously, the second one (B) by direct intra-osseous injection 6 days later. 31% (batch A) and 34.5% (batch B) of the patients CD34 cells expressed gp91 after transduction. There were 1.3 (batch A) and 0.96 (batch B) proviral copies per genome. Following conditioning, we reinfused 2.2 × 10⁶ (batch A) and 3.8 × 10⁶ (batch B) CD34gp91⁺^phox cells/KG resulting in a total dose of 6 × 10⁶ transduced cells/KG. The patient experienced a short period of myelosuppression (Figure 1A). Regular follow-up is ongoing.

Methods
For all NET experiments, neutrophils were resuspended in serum free RPMI media (phenol red-free) supplemented with 10mM Hepes and used within 1 h after isolation. The same media was used to culture A. nidulans conidia and hyphae.

Whole-body PET-CT was conducted according to standard protocols. For immunofluorescence and scanning electron microscopy (SEM) cells were processed as described.⁴

REFERENCES

1. Ott MG, Schmidt M, Schwarzwaelder K, et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med. 2006;12:401–409.
2. Feng Q, Chow PK, Frassoni F, et al. Nonhuman primate allogeneic hematopoietic stem cell transplantation by intraosseus vs intravenous injection: Engraftment, donor cell distribution, and mechanistic basis. Exp Hematol. 2008;36:1556–1566.
3. Frassoni F, Gualandi F, Podesta M, et al. Direct intrabone transplant of unrelated cord-blood cells in acute leukaemia: a phase I/II study. Lancet Oncol. 2008;9:831–839.
4. Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular traps kill bacteria. Science. 2004;303:1532–1535.

Files in this Data Supplement:

Figure S1. NET antifungal activity on A. nidulans conidia and hyphae (JPG, 2.63 MB) -
NET control of A. nidulans conidia (A–H) and hyphae (I–P). Conidia were plated on FACS-sorted neutrophils stimulated with PMA for 4 h, i.e. until NET formation was complete, cells were dead and therefore incapable of phagocytosis. Hyphae were co-incubated with FACS-sorted neutrophils and PMA. Germination and growth of conidia were analyzed after 16 h incubation at 37°C; growth of hyphae after 5 h, revealing NET antifungal activity of control (A, I) and CGD gp91^phox+ (B, J) neutrophils. This activity was significantly reduced after digestion of NETs with Micrococcal Nuclease (MNase) (E, M and F, N). CGD gp91^phox− neutrophils were inefficient in controlling fungal growth (C, K). A. nidulans growth without addition of cells (D–P). A. nidulans survival was visualized as hyphal outgrowth and pictures were taken in parallel to the other experiments shown in Figure 2 using an inverted light microscope coupled to a CCD camera. All experiments were done at MOI 0.5 (A. nidulans: neutrophils) and were repeated at least three times with similar results.
Figure S2. NET independent inhibition of A. nidulans hyphal growth (JPG, 98.6 KB) -
Hyphal outgrowth was measured after 16 h pre-incubation of conidia in media. Hyphae were co-incubated with FACS-sorted neutrophils and PMA ± MNAse and hyphal viability was assessed after 2h (A. nidulans, before NETs formation) at 37°C, revealing NADPH oxidase independent growth inhibition during this time. The data shown are presented as mean ± SD of a representative triplicate experiment. Inhibition of fungal growth is expressed as percentage of control values (A. nidulans hyphae incubated in media).