# Presentation: 728615

## Adenosine-Deaminase (ADA) Deficiency

**ADA is responsible gene in ~20% SCID.**
**Often fatal, if untreated, due to infections.**
**It was the first form of SCID where:**
**	****1. genetic cause was identified (1972),**
**	****2. responsible gene was cloned (1983),**
**	****3. gene therapy was approached (1990),**
**	****4. effective treatment (PEG-ADA) other**
**	****    ****than HSCT was developed (1990).**
**PEG-ADA enzyme replacement therapy: **
**	****1. FDA approved orphan drug (1990),**
**	****2. Bi-weekly I.M.,**
**	****3. Can restore, sustain immunity,**
**	****4. Expensive ($200-500,000/yr).**

## ENZYME REPLACEMENT THERAPY WITH BOVINE ADA (PEG-ADA)

- Correction of metabolic abnormalities.
- Variable restoration of immune functions, with 20% non responders and >50% still on IVIG.
- Last survey (Hershfield, ESID 2002) overall survival 83% (n=113) (73% including patients who underwent BMT).
- 10% developed neutralizing antibodies.
- Autoimmune syndromes in 5 patients (fatal in 3).** **

## Absolute CD3+ T Lymphocyte Counts 

**In ****9**** ADA (-) SCIDs on PEG-ADA 4-11 Yrs**

- Chan ...Kohn
- MS in Prep.

## HLA-identical sibling BMT (treatment of choice)
Survival 75-90%. Neurological and behavioral alterations observed in the long term follow-up.

Non HLA-identical  BMT
Without conditioning (haplo): 33% engraftment  (n=15) (Buckley et al., presented at ESID 2002). 
With conditioning: overall survival 23% (n=29) (EBMT/ESID registry, Antoine et al., Lancet, 2003, 361:553-560).

Overall survival at Great Ormond Street Hospital (B. Gaspar/A. Thrasher), presented at EBMT,  2004
HLA-id sibling/family donor (84%) (n=13)
Matched unrelated donor  or UCB (50%) (n=4)
Haploidentical donor (23%) (n=13)

- Survival 75-90%. Neurological and behavioral alterations observed in the long term follow-up.
- Non HLA-identical  BMT
- Without conditioning (haplo): 33% engraftment  (n=15) (Buckley et al., presented at ESID 2002).
- With conditioning: overall survival 23% (n=29) (EBMT/ESID registry, Antoine et al., *Lancet, 2003, 361:553-560).*
- Overall survival at Great Ormond Street Hospital (B. Gaspar/A. Thrasher), presented at EBMT,  2004
- HLA-id sibling/family donor (84%) (n=13)
- Matched unrelated donor  or UCB (50%) (n=4)
- Haploidentical donor (23%) (n=13)

- Bone Marrow Transplantation for ADA-SCID** **

**Notes:**
But there is also a strong therapeutic rationale compared to other available options, 
In the absence of an HLA-identical sibling domor, an event which occurs in the majority of cases,
BMT is associated with a high risk and poor survival

Enzyme replacement therapy can be an effective but in many patients the immune reconstitution is incomplete and the patients may develop autoimmunity or neutralizing antibodies.

Based on the initial studies which showed the feasbility and safety of gene therapy, but had limited therapeutic impact since the enzyme replavement therapy was maintaiend in all treated patients, we designed an improved clinical gene therapy trial is based

## 
ADA-SCID

	MUD + haploidentical 		23%

SCID T-B+ (including X-SCID)

MUD 				66%
Haploidentical 			50%

**ADA-SCID**
**	**
**	****MUD + haploidentical** 		**23%**
**SCID T-B+ (including X-SCID)**
**MUD ****	****	****	****	****66%**
**Haploidentical ****	****	****	****50%**

- Survival after HLA-mismatched
- Bone Marrow Transplantation for SCID** **
**(EBMT/ESID registry, Antoine et al., *****Lancet, 2003, 361:553-560)***

**Notes:**
But there is also a strong therapeutic rationale compared to other available options, 
In the absence of an HLA-identical sibling domor, an event which occurs in the majority of cases,
BMT is associated with a high risk and poor survival

Enzyme replacement therapy can be an effective but in many patients the immune reconstitution is incomplete and the patients may develop autoimmunity or neutralizing antibodies.

Based on the initial studies which showed the feasbility and safety of gene therapy, but had limited therapeutic impact since the enzyme replavement therapy was maintaiend in all treated patients, we designed an improved clinical gene therapy trial is based

## Early ADA Gene Therapy Trials

**	****	****	****	****	****	****	**_**# of patients**_
_**T cells**_
- Blaese et al. 1993				2
- Bordignon et al. 1992				6*
_**CD34+ cells**_
- Bordignon et al. 1992			         2*
- Hoogerbrugge et al. 1992			3
- Kohn et al. 1993					3
*** same patients**

## 1st CHLA/NIH ADA Gene Transfer Trial 

- In 1993, umbilical cord blood was collected from
- three ADA-deficient SCID neonates.
- CD34+ cells were isolated and transduced with
- the human ADA cDNA by culture for 3 days with
- the LASN retroviral vector and IL-3/IL-6/SCF.
- The cells were reinfused I.V. on day 4 of life,
- without prior cytoreduction.
- Treatment with PEG-ADA was initiated.

## Frequency of Gene-Containing Leukocytes Measured Using Semi-Quantitative PCR

**Months after birth**

_**UPN #ADA101**_
**X=gran;      = PBMC;**
**M=monocytic; **
**T= T cell; B= B cell**

**Kohn et al, *****Nat Med***** 4:775-780, 1998**.

**PEG-ADA (U/kg/wk)**

## LAM-PCR analysis of PBMC, T cells and myeloid cells

**From: Schmidt et al., Nat Med. 2003; 9(4):463-8**

## Summary Schmidt et al., Nat Med. 2003; 9:463-8

-   LAM-PCR revealed the stable presence of a predominant
- vector integrant in T and myeloid cells over the past 8 years.
-    T cell clones grown from peripheral blood 8 years after
- neonatal CD34+ cell gene transduction indicated that:
-    	a single pre-thymic stem or progenitor cell accounted
- 	for the majority of gene marking in  polyclonal T cell
- 	production.

## Frequency of Gene-Containing Leukocytes Measured Using Semi-Quantitative PCR

**Months after birth**

_**UPN #ADA101**_
**X=gran;      = PBMC;**
**M=monocytic; **
**T= T cell; B= B cell**

**Kohn et al, *****Nat Med***** 4:775-780, 1998**.

**PEG-ADA (U/kg/wk)**

**X**

**+11 yrs**
**       ****↓**

**       ****↑**
**+11 yrs**

## 2nd CHLA/NIH ADA Gene Transfer Trial

- 1. Phase 1 study
- 2. 10 patients - must be on PEG-ADA E.R.T.
- 3. ADA-deficient SCID neonates or children
- 4. Target cell: CD34+ cells from UCBC (neonates) or BM (children)
- 5. Gene transfer method: *Ex vivo* transduction with MLV-based RV in GALV-pseudotype using
- 	SCF/MGDF/F3L on retronectin, serum-free.
- 6. Phased withdrawal of PEG-ADA after 1 year,
-   	 if gene marking present.
- 7. 2 year active phase follow-up.

**2****nd**** ****C****H****L****A****/****N****I****H**** ****ADA ****Gene Transfer Trial**

## 2nd CHLA/NIH ADA Gene Transfer Trial

**IND Application, Aug. 1999**

**IND Approval 2001**

**4 patients enrolled, Aug 2001 – Jan 2002**

- _UPN__	__	__Age (y/o)__	__CD34+/kg__	__% PCR+ CFU_
- 201C		15		    0.7			12*
- 202N		  5		  13.3			50
- 203N		20		    1.3			  1
- 204C		  4		    2.0			20
- * GcSap vector only

## ADA Vector Marking

**Months Post-Infusion **

**# Proviral Copies / Cell**

**15 y/o**

**5 y/o**

**20 y/o**

**  ****4 y/o**

##   Clinical Trial of Gene Therapy for ADA-Deficient SCID in Italy

- Aiuti et al. (Milan).  Science 296:2410-2413, 2002.
- Two ADA-deficient SCID given busulfan (4/kg) prior to BM infusion (“non-myeloablative conditioning”).
- Not treated with PEG-ADA therapy.
- Immune reconstitution by 6 months.
- 	T cells gene-marked at 100%
-  	Myeloid cells gene-marked at 7-12%.
- ----------------------------------------------------------------------
- 4 more treated since then, with good immune recovery

## ADA-SCID gene therapy: the Milan trial 

**ADA-SCID gene therapy: the ****Milan**** trial **

***(Aiuti et al. Science, 2002, 296:2410-3 and unpublished data)***

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## T-cell Reconstitution in early phase: 

**T-cell ****R****econstitution in early phase: **
**comparison of SCID trials**** **

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***(******Hacein-Bey et al. Science, ***
***2003, 302:415-9)***

***(Aiuti et al. Science, 296:2410-3 ***
***2002 and  unpublished data)***

**ADA-SCID**

**X-SCID**

**T cells/microl**

- Months of follow up

**X****SCID**

## 2nd CHLA/NIH ADA Gene Transfer Trial

**IND Application, Aug. 1999**

**IND Approval 2001**

**4 patients enrolled, Aug 2001 – Jan 2002**

**Clinical Hold, Sep. 2002**

**Clinical Hold lifted Dec 2003**

**IND changes, incl. Busulfan, PEG-ADA withdrawal, age and cell dose limit, final approval:**** ****Jan 2005**

**Clinical Hold, Jan. 2005**

## ADA (-) SCID: Summary

- PEG-ADA palliative, but immune function is below normal
- Poor outcome with haplo-BMT
- No adverse events in at least 18 subjects,   some with retroviral-transferred gene present >10 years
- Good outcome from gene therapy in Milan study, using Busulfan and no PEG-ADA