Morpholino-based correction of hypomorphic ZAP70 mutation in an adult with combined immunodeficiency.

To the Editor:

The ζ-associated protein of 70 kDa (ZAP-70) is a cytoplasmic tyrosine kinase critical for intracellular signaling downstream of the T-cell receptor (TcR). Biallelic mutations in ZAP70 cause combined immunodeficiency (CID). Classically, ZAP-70 deficiency results from null alleles and presents with failure to thrive and severe/recurrent infections in infancy (ie, severe CID) that is marked by absent circulating CD8+ T cells, quantitatively normal yet dysfunctional CD4+ T cells accounting for hypo-/dysgammaglobulinemia, with quantitatively intact B cells and natural killer cells and requires hematopoietic stem cell transplant for survival beyond early childhood1, 2, 3, 4, 5 (see Table E1 in this article's Online Repository at www.jacionline.org). “Leaky” ZAP-70 deficiency has been reported only once: a 9-year-old boy with nonsevere skin and lung infections had a homozygous intronic mutation producing a novel splice acceptor site, resulting in predominant expression of a frame-shifted mutant protein but with residual levels of wild-type (WT) protein. Recently, a sibling pair manifesting only early-onset autoimmune diseases, without opportunistic infections, was shown to be compound heterozygous for a hypoactive allele and a weakly hyperactive allele; the specific combination of these paired alleles was required for disease. Collectively, these cases show that residual levels of functional protein regulate the clinical phenotype of ZAP-70 deficiency. It further suggests that, in distinction to null alleles, different therapeutic approaches may be needed for “leaky” mutations that permit survival beyond the typical time frame and for whom hematopoietic stem cell transplant at older age may be associated with excess risk. We expand the clinical phenotype of hypomorphic ZAP-70 deficiency and provide proof-of-concept that mutation-targeted therapy can improve immune function.

Table E1

Reports of ZAP-70 deficiency

Case	Reference	Mutation	Effect on protein	Age of onset; sex	Infections	Autoimmune/dysregulated inflammation	Immunologic phenotype	Outcome
1	E15, E16, E17	Compound heterozygous		<1 y-old M; Mennonite	Upper respiratory tract Infections; Pneumonia; Oral ulcerations; PCP*	Diarrhea, weight loss	Total CD3: normal.CD4+ T: normal.CD8+ T: absent.B: elevated.Immunoglobulin: IgG (↑); IgA (↑); IgM (N); IgE (NR)	HLA-matched unrelated BMT at 20 mo with successful engraftment but died at 22 mo (unclear etiology)
		c.1763C>A	p.S518R.No protein detected in patients' cells.
		c.1624-11G>A [NM_001079.3]; G to A transition in intron 12 [g.98354447G>A; assembly GRCh37]	Creation of stronger splicing acceptor site leading to addition of 3 amino acids (LEQ) in the catalytic domain. No protein detected in patients' cells
2	E15, E16, E17			Neonate M (brother of above case); identified by immunophenotypic screen; Mennonite	None (kept in protective isolation).(Had CMV in urine and rotavirus in stool, but was asymptomatic)	NR	Total CD3: decreased.CD4+ T: normal.CD8+ T: absent.B: elevated.Immunoglobulin: NR	HLA-matched unrelated BMT at 4 mo; alive at age 2 y
3	E15, E16, E17			6 mo F; Mennonite; unrelated to above cases	Otitis media with perforation; PCP	NR	Total CD3: normal.CD4+ T: elevated.CD8+ T: absent.B: normal.Immunoglobulin: IgG (↓); IgA (↑); IgM (N); IgE (NR)	HLA-matched related BMT at 11 mo; alive at 4 y
4	E15, E16, E17, E18			5 mo F (sister of case 3); Mennonite; identified by immunophenotypic screen	Oral ulcerations with eczematous rash; PCP; chronic diarrhea with reovirus; oral thrush	NR	Total CD3: normal.CD4+ T: elevated.CD8+ T: absent.B: normal.Immunoglobulin: IgG (N); IgA (N); IgM (N); IgE (NR)	Partial HLA-matched unrelated BMT at age 24 mo; alive at 3 y
5	E19	Homozygous: 1719_1931del13 with predicted frameshift	No protein detected in patient's cells	NR	NR	NR	CD4+ T: normal.CD8+ T: absent.B: normal.Immunoglobulin: NR	NR
6	E20, E21	Mutation not defined but no mRNA detected	No protein	4.5 mo M; not Mennonite	Multiple respiratory infections; laryngomalacia; enteritis due to coronavirus; pneumonitis due to human parainfluenza virus 3	Diarrhea, failure to thrive	CD4+ T: normal.CD8+ T: absent.B: elevated.Immunoglobulin: pan-hypo-gamma globulinemia	BMT at 7 mo from mother with survival
7	E22, E23	Compound heterozygous		2 mo M; Japanese	None (kept in isolation because of skin lesions in sister, who died of CMV pneumonia at 6 mo)	Infiltrative skin lesions on face & extremities; No diarrhea	Total CD3: NR.CD4+ T: NR.CD8+ T: absent.B: normal.Immunoglobulin: IgG (N); IgA (N); IgM (N); IgE (↑)	Well at 17 mo
		c.C448A	p.P80Q
		c.A1923T	p.M572L
			Temperature-sensitive instability of proteins: degradation at 37°C; detectable protein at 30°C
8	E24	Homozygous: 1719_1931del13 with predicted frameshift	No protein detected in patient's cells	1-y-old F (unrelated to case 5)	PCP at age 3 mo; extensive varicella infection at 6 mo	NR	Total CD3: NR.CD4+ T: NR.CD8+ T: absent.B: normal.NK: normal.Immunoglobulin: NR	NR
9	E25	Homozygous: c.C1729T	p.A507V.No protein detected in patient's cells	2 siblings NOS	NR	NR	Total CD3: NR.CD4+ T: normal.CD8+ T: severely low (<3%).B: NRImmunoglobulin: NR	NR
10	E26	Homozygous: c.C1602T	p.R465C. This mutation does not impair ZAP-70 expression or autophosphorylation but it impairs its kinase activity	10 mo M; white	PjP at 7 mo	NR	Total CD3: NR.CD4+ T: NR.CD8+ T: few (<170 cells/μL).B: normal.Immunoglobulin: IgG (↓); IgA (N); IgM (N); IgE (NR)	T-cell–depleted BMT from mother at 1 y, complicated by non-EBV large B-cell lymphoma that was treated. Subsequently underwent peripheral SCT from father
11	E27	Homozygous: g.G1603A	p.R465H	8 mo F; NOS	Recurrent respiratory tract infections	NR	Total CD3: NR.CD4+ T: NR.CD8+ T: absent.B: NR.Immunoglobulin: NR	Haplo-identical HSCT (from father) at 9 and 10 mo, but engraftment not successful
12	E28	Homozygous: 836+121G>A	Creation of a new splicing acceptor site in intron 7 (78 bp upstream of the normal splice site), creating an in-frame product with a stop codon 61 bp after the new splice site. Decreased expression of WT protein (hypomorphic)	9 y M; NOS	Skin infections; lung infections; no opportunistic infections	No autoimmunity. No lymphoproliferative disease	Total CD3: ↓.CD4+ T: ↓.CD8+ T: ↓.B: normal.Immunoglobulin: IgG (N); IgA (N); IgM (N); IgE (↑)	Alive
13	E29	Homozygous: c.1520C>T	p.A507V; absence of protein detection	4 mo F	Recurrent lower respiratory tract infections	None reported	Total CD3: N.CD4+ T: N.CD8+ T: ↓.B: NR.Immunoglobulin: IgG (N); IgA (↑); IgM (N); IgE (NR)	Awaiting HSCT at time of reporting
		Homozygous: c.1010T>G	p.L337R; absence of protein detection	3 mo F	Recurrent gastroenteritis. Recurrent lower respiratory tract infections.Oral candidiasis. Failure to thrive	Secondary hemophagocytic syndrome	Total CD3: ↓.CD4+ T: N.CD8+ T: ↓.B: NR.Immunoglobulin: IgG (↑); IgA (N); IgM (↑); IgE (NR)	Died of multiorgan failure awaiting HSCT
		Homozygous: c.1690T>C	p.C564R; absence of protein detection	2 mo F	Recurrent pneumonia.Oral candidiasis.Probable BCGosis	Exfoliative dermatitis. Subcutaneous nodules (probable BCGogsis).Generalized erythodermia with eosinophilia (Omenn)	Total CD3: ↑.CD4+ T: ↑.CD8+ T: ↓.B: NR.Immunoglobulin: IgG (↑); IgA (↑); IgM (N); IgE (NR)	Awaiting HSCT at time of reporting
14	E30	Homozygous c.836_837delAT	Absence of protein detection	11 mo F	EBV-associated diffuse large B-cell lymphoma	NR	Total CD3: N.CD4+ T: N.CD8+ T: ↓ (<1%).B: N.NK: N.Immunoglobulin: (NR)	Died of pneumonitis, disseminated intravascular coagulation, and multiorgan failure
15	E31	Homozygous: c.C1153T	p.R385C	5 mo M; Turkish (sibling 1)	PjP at 5 mo	Cholestatic liver disease due to toxic cholangitic hepatitis with portal fibrosis	Total CD3: (NR).CD4+ T: (NR).CD8+ T: (NR).B: (NR).NK: (NR).Immunoglobulin: (NR)	BMT from matched, unrelated female donor at 8 mo. Required liver transplant 2 y after BMT.Alive
				4-wk-old M; Turkish (sibling 2)	NR	NR	Total CD3: N.CD4+ T: N.CD8+ T: absent.B: N.NK: N.Immunoglobulin: (NR)	Combined transplant: CD34+ stem cells from same donor of sibling 1, plus peripheral blood lymphocytes of sibling 1
16	E32	Homozygous c.C1747>T	p.R514C	5 mo F; Turkish	Pneumonia	Icthyosiform skin rash at 2 mo.Severe erythrodermia at 5 mo.High IgE	Total CD3: N.CD4+ T: N.CD8+ T: ↓.B: N.NK: ↓.Immunoglobulin: IgG (N); IgA (N); IgM (N); IgE (↑)	Died because of CMV pneumonitis (age NR)
17	E33	Compound heterozygous		9 mo M; 1 mo F; white	None	9 mo M:Nephrotic syndrome (minimal change disease); bullous pemphigoid (20 mo); acquired hemophilia from autoantibody to factor VIII (2 y); inflammatory colitis (3 y)1 mo F: Bullous pemphigoid; inflammatory colitis (NOS); nephrotic syndrome (NOS)	9 mo M:Total CD3: ↓.CD4+ T: (NR).CD8+ T: ↓.B: ↓.NK: (NR).Immunoglobulin: IgG (↓); IgA (NR); IgM (NR); IgE (NR)1 mo F:Total CD3: (NR).CD4+ T: (NR).CD8+ T: ↓.B: ↑.NK: (NR).Immunoglobulin: (NR)	Male: HSCT at 5 y with resolution of autoimmune manifestationsFemale: HSCT at 6 mo with resolution of pemphigoid; HSCT at 28 mo because of declining donor chimerism and autoimmune hypothyroidism, with resolution of disease
		c.574C>Tc.1079G>C	p.R192Wp.R360P

BCGosis, Disseminated BCG infection; BMT, bone marrow transplant; CMV, cytomegalovirus; F, female; HSCT, hematopoietic stem cell transplant; M, male; NK, natural killer; NOS, not otherwise specified; NR, not reported; PCP, Pneumocystis carinii pneumonia; PjP, Pneumocystis jirovecii pneumonia; SCT, stem cell transplant.

We identified a 33-year-old male (P1) born from consanguineous Coptic parents (see Fig E1, A, in this article's Online Repository at www.jacionline.org). An older brother died at 16 months from suspected viral encephalitis; an older sister and parents were well. P1 had a history of failure to thrive, adrenal insufficiency, and inflammatory enterocolitis starting in infancy, recurrent respiratory tract infections starting at 9 months, and intermittent mucocutaneous candidiasis, recurrent herpes simplex virus stomatitis, and varicella-zoster virus infections 3 times. Investigations at the time revealed a visible thymus, normal immunoglobulin isotypes with poor response to vaccines, and persistent CD8+ lymphocytopenia (1% to 3% of total T cells age-adjusted reference range). Beginning at approximately 3 years of age, serial testing demonstrated increasing proportion of CD8+ T cells (8% to 10% of total lymphocytes). Further investigations were suggestive of ZAP-70 deficiency (Fig E1, B and C). However, a reference laboratory reported identifying no mutation in ZAP70. Thus, bone marrow transplantation was not pursued. IgA nephropathy at age 16 years eventually required renal transplant from his mother at age 30 years. The subsequent years were marked by EBV viremia/lymphoproliferative disorder (with recurrence of viremia ∼6 months after completing rituximab), cytomegalovirus viremia (with recurrence following cessation of (val)ganciclovir), polyomaviremia (BK and JC viruses), and epidermodysplasia verruciformis-like lesions (due to human papilloma virus-23) (see Fig E2 in this article's Online Repository at www.jacionline.org), which prompted reevaluation for an underlying immunodeficiency. He is currently aged 35 years, on immunoglobulin replacement for respiratory tract infection prophylaxis, and his viral infections are managed conservatively.

Fig E1

A, P1 pedigree. An older brother had a papulo-squamous rash at age 1 month, recurrent otitis media, and died at 15 months following recurrent seizures, suspected to be viral encephalitis by imaging, electroencephalogram, and autopsy. B, During P1's first 2 years of life, summaries of investigations revealed decreased CD8+ T lymphocytes with very low T-cell mitogen (PHA and concanavailin A) proliferative responses. B lymphocytes were quantitatively normal with preserved proliferation in response to pokeweed mitogen. Circulating immunoglobulin concentrations (IgG, IgA, IgM) were normal, with IgE occasionally being slightly elevated. Subsequent investigations found no evidence of adenosine deaminase (ADA) or purine nucleoside phosphorylase (PNP) deficiency with normal expression of HLA on B cells. C, T-cell lysates from P1 and a healthy control (HC) were analyzed by immunoblot with indicated antibodies.

Fig E2

A, Well-circumscribed pink macules on the trunk torso. B, Hematoxylin and eosin stain demonstrating enlarged superficial keratinocytes with distinctive blue-gray cytoplasm in association with prominent keratohyaline granules, consistent with HPV infection. C, Immunohistochemical pan-HPV stain.

Whole-exome sequencing revealed no known etiology for the immunodeficiency, other than a homozygous c.1272C>T variant in ZAP70 (NM_001079; hg19). The germline c.1272C>T variant, while synonymous (p.G355G), is predicted in silico (Human Splicing Finder) to create a novel donor splice site within exon 9 (DSSmut) that is stronger than the native junctional one (DSSwt) (Fig 1 , A). This new splice site is predicted to create a 19-bp deleted product with a premature stop codon in exon 10. Sanger sequencing confirmed the homozygous c.1272C>T variant in P1; the unaffected family members were heterozygous (Fig 1, B). RT-PCR from P1's PBMCs confirmed the predicted splicing effect, demonstrating a predominant mutant isoform with trace levels of WT isoform (Fig 1, C and D). The heterozygous family members possessed both isoforms but with higher WT levels; unrelated healthy controls expressed only the WT isoform. Sequencing of gel-eluted bands confirmed the 19-bp deletion in the mutant isoform (data not shown). Thus, c.1272C>T variant creates a mutant mRNA isoform through aberrant splicing; in homozygous state, the mutant isoform predominates over WT in P1.

Fig 1

A, The c.1272C>T ZAP-70–altered splice site. B, Electropherograms from a healthy control (HC), P1, and P1's family. C, RT-PCR products from an HC, P1, and P1's family. D, Summary of densitometry analysis. E, Immunoblot of ZAP-70wt or ZAP-70mut transfected cells. F, The AMO binding site within the ZAP-70 pre-mRNA. An asterisk denotes the c.1272C>T mutation. G, RT-PCR products of AMO-treated P1 PBMCs.

To determine the impact of the predominant aberrant ZAP70 mRNA isoform on protein production, we performed immunoblot analysis on P1's PBMCs and confirmed reduced ZAP-70 levels, as was seen in the original childhood investigations (Fig E1, C). Plasmids encoding the open-reading frame of WT ZAP-70 (ZAP-70wt) or the 19-bp deleted variant (ZAP-70mut) were stably transfected into ZAP-70–deficient Jurkat P116 cells, and immunoblot of cell lysates confirmed the absence of ZAP-70 protein with the mutant isoform (Fig 1, E). TcR signaling downstream of ZAP-70, assessed by phosphorylation of linker for activation of T cells, was absent following OKT3 stimulation of ZAP-70mut–transfected Jurkat P116, but intact with ZAP-70wt (see Fig E3, B, in this article's Online Repository at www.jacionline.org). These findings confirm that the mutant ZAP70 mRNA resulting from the c.1272C>T variant is null.

Fig E3

A, A schematic representation of the ZAP-70 protein, with the asterisk denoting the location of the c.1272C>T mutation. B, Jurkat P116 cells were stimulated with OKT3 antibody and cell lysates were analyzed by immunoblot using antibodies specific for LAT and phospho-LAT. LAT, Linker for activation of T cells.

Although the mutant donor splice site (DSSmut) is stronger than the native WT site (DSSwt), the latter is still intact and functionally able to generate WT ZAP70 mRNA and protein. In P1's homozygous mutant state, this is clearly inadequate for T-cell homeostasis. However, in heterozygous family members, ZAP-70 WT protein is sufficiently produced to maintain an otherwise well state. We hypothesized that blocking the stronger DSSmut in P1 would favor usage of DSSwt to increase WT ZAP-70 levels and consequently, T-cell function. To prove this, we designed an antisense morpholino oligonucleotide (AMO) to target the c.1272C>T variant while sparing the native exon-intron junction so as to block the mutant splice effect during processing of ZAP70 pre-mRNA (Fig 1, F). To confirm that the AMO specifically inhibited expression of the mutant ZAP70 isoform (ie, blocked its transition from pre-mRNA to mRNA), we treated P1's PBMCs and demonstrated correction of splicing (Fig 1, G). Optimization studies demonstrated maximal corrective effect starting at 48 hours using 5 μM AMO, producing approximately 50% WT mRNA (confirmed by sequencing; data not shown). A similar AMO-induced effect on mutant mRNA was seen on the sister's cells (data not shown); there were insufficient number of cells from P1's parents for testing. To verify the specificity of this effect, we treated cells from healthy controls and observed no effect (data not shown). Immunoblot analysis of P1's AMO-treated PBMCs demonstrated increased ZAP-70 expression, confirming that the correction of aberrant splicing results in augmented production of ZAP-70 protein (Fig 2 , A and B). AMO-treated PBMCs demonstrated increased linker for activation of T cells activation following OKT3 stimulation compared with untreated cells (Fig 2, A and B), indicating that this restored ZAP-70 retains functionally intact TcR signaling. To determine whether the AMO could improve cellular function, we evaluated the expression of prototypical T-cell cytokine genes, IL2, IL4, IL10, and IFNG (IFN-γ) (Fig 2, C). AMO treatment alone increased IL2, IL4, and IL10 expression; IL2 and IL4 expression was further enhanced by CD3/CD28 stimulation (Fig 2, C). Treatment with AMO had no effect on IFN-γ expression (data not shown), likely due to the reestablished TH2-driven responses in the PBMCs polarizing away from TH1 responses. Last, AMO treatment reestablished the proliferative response to CD3/CD28 costimulation in P1's primary T cells, an effect seen in both CD4+ and CD8+ T cells (Fig 2, D and E; see Fig E4 in this article's Online Repository at www.jacionline.org). Collectively, these findings demonstrate that AMO treatment increases the expression of WT ZAP-70 protein, which is molecularly intact and capable of restoring key T-cell functional responses.

Fig 2

A, AMO-treated PBMCs from a healthy control (HC) or P1 stimulated with OKT3, with densitometry analysis summarized in B. C, AMO-treated HC or P1 PBMCs stimulated with CD3/CD28 as indicated. D, CFSE profiles of HC or P1 CD3/CD28-stimulated T cells, with percentages summarized in E. One experiment representative of at least 2 independent experiments is shown. CFSE, Carboxyfluorescein succinimidyl ester. *P < .05.

Fig E4

Gating strategy for assessment of T-cell proliferation of CFSE-labeled, CD3/CD28-stimulated cells. APC, Allophycocyanin; CFSE, carboxyfluorescein succinimidyl ester; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Analysis of this hypomorphic ZAP-70–deficient patient uniquely illustrates several key points: (1) Clinically, the morbid natural history suggests a role for ZAP-70 in the functional control of double-stranded DNA viruses beyond the generation of CD8+ T cells, which requires further elucidation. Interestingly, the autoimmune diseases here (colitis; nephropathy) were also reported by Chan et al, who elegantly demonstrated that a fine molecular balance in ZAP-70 effect on T-cell signaling can be associated with autoimmunity only. (2) Genetically, it shows that synonymous genetic variants may be deleterious by modifying splicing, with hypomorphism resulting from a skewed net balance of amorphic versus residual WT protein. (3) Immunotherapeutically, it demonstrates that the accurate identification of causal genetic lesions permits a mutation-silencing approach that leads to molecular correction and recuperation of fundamental T- cell cytokine responses, at least in vitro. Although the breadth of T-cell functions could not be fully investigated pragmatically, to our knowledge this represents the first example in which AMO therapy successfully restored immune function in primary cells from a human with a genetically defined immunodeficiency. This approach could also extend to therapeutically modulate functional single-nucleotide polymorphisms in immune genes. Clearly, additional parameters will need to be addressed before this approach can be used in clinical practice (eg, pharmacokinetics and safety), although this strategy is in use for inborn errors of muscle or metabolism. Overall, the insight gained from this work opens new avenues for individualized, mutation-targeting therapy for CID.