Gene Therapy - Clinical Tree

Introduction

In recent years, the number of gene therapy (GT) trials targeting the liver have grown significantly, particularly when it comes to gene addition strategies (delivery of cDNA for a whole gene to the target organ/cells, which then exists in addition to the host genome with the mutated gene) to rescue monogenic diseases. Although several gene delivery approaches have been tested in preclinical animal models, including viral and nonviral approaches, adeno-associated virus (AAV) vector-based gene transfer remains the most popular gene delivery platform for the liver, with a few trials entering phase III. Excellent safety profile, high efficiency of transduction of hepatocytes, and evidence of long-term efficacy deriving from early-phase trials have established AAV vectors as the platform of choice for the treatment of enzyme deficiencies and metabolic disorders. The large number of proof-of-concept studies in animal models showing therapeutic efficacy following AAV vector gene transfer is evidence of a potentially rich pipeline of GT drugs that could potentially be brought to the clinic and change the way genetic diseases could be treated, particularly liver diseases.

Clinical translation of novel therapies, however, is a process that involves several bench-to-bedside cycle iterations, during which issues associated with the technology are encountered and solved. For the AAV vector gene transfer technology, several hurdles have been highlighted both in preclinical studies and clinical trials; addressing these issues will contribute to expanding the number of indications in which clinical success can be achieved.

In this chapter, we will discuss some of the key approaches to GT for liver-based metabolic diseases (LBMDs), with a focus on the advances of gene addition approaches and AAV-based GT strategies, and will present some of the main achievements, and emerging issues, of the field of in vivo gene transfer. These emerging issues are:

1.
AAV vector and transgene immunogenicity;
2.
genotoxicity risk associated with AAV gene transfer to the liver;
3.
AAV gene transfer persistence in a developing versus adult liver.

Why Gene Therapy? What Is the Advantage Compared With Liver Transplantation? Pros and Cons for Liver Transplantation and Gene Therapy

Inherited diseases are a substantial burden of childhood disease, and they result in up to 70% of all admissions to children’s hospitals. Because most metabolic activity in the body happens in the liver, and furthermore, most metabolic diseases affect, at least partly, the liver, a significant subgroup of inherited diseases is the group of LBMDs. In some conditions, all of the defective or deficient enzymes are expressed in the liver; in others, they are partially expressed in the liver and partially in other organs such as muscle, kidney, or central nervous system ( Table 33.1 ). In many LBMDs, the livers themselves are not diseased ( Table 33.1 ).

Table 33.1

The Most Common Liver-Based Metabolic Diseases and/or Where Liver Transplantation Has Been Reported

	Expression Mainly From Hepatocytes/Only Hepatocyte-Based Pathway	Expression Also in Other Organs/Impaired Pathways in Other Organs
With progressive liver disease/fibrosis - selection advantage for corrected cells - disease dependent up to high risk for HCC	Group A ➢ Wilson disease (1:30,000) ➢ Alpha-1 antitrypsin deficiency (1:2500) ➢ BSEP deficiency ^a ➢ MDR3 gene defect ^a ➢ Tyrosinemia type 1 (1:120,000)	Group B ➢ Cystic fibrosis (1:2400) ➢ Glycogen storage disease ^b types III, IV ➢ FIC1 deficiency ^a ➢ Glycogen storage disease ^b type I (1:100,000)
Without or slow progressive liver disease if treated - no selection advantage for corrected cells - no risk for HCC	Group C ➢ Crigler-Najjar syndrome (1:1,000,000) ➢ Ornithin transcarbamylase deficiency (1:14,000) ➢ Primary hyperoxaluria (1:120,000) ➢ Familial hypercholesterolaemia (1:1,000,000) ➢ Coagulation defects	Group D ➢ Citrullinemia (1:57,000) ➢ Cystinosis (1:100,000) ➢ Propionic acidemia (1:100,000) ➢ Maple syrup urine disease (1:185,000)

Expression Mainly From Hepatocytes/Only Hepatocyte-Based Pathway

Expression Also in Other Organs/Impaired Pathways in Other Organs

With progressive liver disease/fibrosis

-
selection advantage for corrected cells
-
disease dependent up to high risk for HCC

Group A

➢
Wilson disease (1:30,000)
➢
Alpha-1 antitrypsin deficiency (1:2500)
➢
BSEP deficiency ^a
➢
MDR3 gene defect ^a
➢
Tyrosinemia type 1 (1:120,000)

Group B

➢
Cystic fibrosis (1:2400)
➢
Glycogen storage disease ^b types III, IV
➢
FIC1 deficiency ^a
➢
Glycogen storage disease ^b type I (1:100,000)

Without or slow progressive liver disease if treated

-
no selection advantage for corrected cells
-
no risk for HCC

Group C

➢
Crigler-Najjar syndrome (1:1,000,000)
➢
Ornithin transcarbamylase deficiency (1:14,000)
➢
Primary hyperoxaluria (1:120,000)
➢
Familial hypercholesterolaemia (1:1,000,000)
➢
Coagulation defects

Group D

➢
Citrullinemia (1:57,000)
➢
Cystinosis (1:100,000)
➢
Propionic acidemia (1:100,000)
➢
Maple syrup urine disease (1:185,000)

a Cumulative incidence, 1:50,000–100.000.

b Cumulative incidence, 1:20,000. BSEP , Bile salt export protein; HCC, hepatocellular carcinoma.

GT could cure or at least treat inherited diseases and is much more curative than other treatment options so far while having the potential to have fewer side effects and burden for the patient compared with liver transplantation (LT). In addition, LT treatment is often limited to supportive measures and may entail significant adverse effects, long-term organ failure, risk of metabolic crises, and malignancy or impairment in the quality of life. Especially for LBMDs without structural damage in the liver ( Table 33.1 , group C and D), the LT is a rough kind of “gene correction.” Even though LT for LBMDs has an excellent long-term outcome, with 5-year survival above 90%, the procedure is associated with significant morbidity and mortality. The benefits and risks of GT compared with LT are different depending on disease and disease group, corresponding to Table 33.1 and like those shown in Table 33.2 . In Table 33.3 , general advantages and disadvantages of GT (exemplary for nonintegrating or targeted integrating recombinant AAV [rAAV] GT approaches) versus LT are shown.

Table 33.2

Disease Group–Specific Aspects for Gene Therapy

	Expression Only From Hepatocytes/Only Hepatocyte-Based Pathway	Expression Also in Other Organs/Impaired Pathways in Other Organs
With progressive liver disease/ fibrosis - selection advantage for corrected cells - disease dependent up to high risk for HCC	Group A ➢ In most diseases, high risk for HCC, even though the metabolic phenotype is cured by GT ➢ Phenotype is easy to cure because of selection advantage	Group B ➢ GT curing disease in all organs would be a great success ➢ Especially because LT for these diseases is only a partial phenotype correction and, in some cases, LT can generate new disease-associated problems ➢ Gene therapy difficult to perform because different organs have to be targeted ➢ Lower risk for HCC
Without progressive liver disease - no selection advantage for corrected cells - no risk for HCC	Group C ➢ Most suitable diseases for GT ➢ No problems with uncorrected cells	Group D ➢ Also very suitable for GT, but efficient phentoype correction is more difficult because of gene expression also outside the liver

Expression Only From Hepatocytes/Only Hepatocyte-Based Pathway

Expression Also in Other Organs/Impaired Pathways in Other Organs

With progressive liver disease/ fibrosis

-
selection advantage for corrected cells
-
disease dependent up to high risk for HCC

Group A

➢
In most diseases, high risk for HCC, even though the metabolic phenotype is cured by GT
➢
Phenotype is easy to cure because of selection advantage

Group B

➢
GT curing disease in all organs would be a great success
➢
Especially because LT for these diseases is only a partial phenotype correction and, in some cases, LT can generate new disease-associated problems
➢
Gene therapy difficult to perform because different organs have to be targeted
➢
Lower risk for HCC

Without progressive liver disease

-
no selection advantage for corrected cells
-
no risk for HCC

Group C

➢
Most suitable diseases for GT
➢
No problems with uncorrected cells

Group D

➢
Also very suitable for GT, but efficient phentoype correction is more difficult because of gene expression also outside the liver

GT, Gene therapy; HCC, hepatocellular carcinoma; LT, liver transplantation.

Table 33.3

Comparison of rAAV Gene Therapy With Hepatic Transplantation for Liver-Based Metabolic Diseases

	Gene Therapy	Liver Transplantation
Concept	Only correcting defect gene in healthy liver	Replacement of a healthy liver to correct one gene
Experience, knowledge	Less experience, unknown long-term outcome	Established treatment, good long-term outcome
Safety	Risk of insertional mutagenesis, off-target effects	Perioperative mortality, mortality on waiting list
Efficiency	Not all hepatocytes will be corrected; correction of some hepatocytes is usually sufficient for phenotypic correction	All hepatocytes were replaced
Stability/persistence	Depending on the approach and age of the patient	Usually lifelong, but need for liver retransplantation because of chronic rejection or other problems is possible
Patient selection	So far, only feasible in patients without preexisting antibodies (new approaches to circumvent this are under progression/evaluation). Problems with stability/persistence in patients under 10 years.	Feasible at all ages, but risk of mortality, morbidity, and long-term side effects of immunosuppression
Immunosuppression	No or short-time immunosuppression	Lifetime immunosuppression with side effects such as elevated risk for tumors and kidney dysfunction
Transgene antibodies	Many studies have shown that rAAV-based liver-directed gene therapy causes immunological acceptance of transgene	Evident in some diseases, for example, BSEP deficiency (PFIC2)
Application	Portal vein or peripheral vein vector infusion	Substantial surgery
Resources	Unlimited resources	Organ shortage
Timing	Therapy at every time, no waiting list	Cannot be influenced, depending on organ offers

Seroprevalence for AAV8, 20%–30%; for AAV2, 50%–60%.

BSEP, Bile salt export protein; PFIC2, progressive familial intrahepatic cholestasis type 2; rAAV, recombinant adeno-associated virus.

For diseases with cell-autonomous behavior like primary hyperoxaluria and/or defects not only evident in the liver ( Table 33.1 , groups B and D), for example, propionic academia, GT is more difficult, especially when the aim is to transduce the other organs also, but at the same time it has the potential to deliver a treatment that is much closer to cure than LT, where only the defect in the liver will be treated. The first promising results for this are shown in propionic academia.

The Clinical Experience With Liver Gene Transfer

The liver is a particularly attractive organ for the development of gene-based therapeutic approaches because it presents several attractive organ-specific features, which include (1) the fact that it is one of the body’s major biosynthetic organs, which confers an advantage when it comes to turning hepatocytes into biosynthetic units; (2) the liver has a unique dual blood supply from the hepatic portal vein and hepatic arteries and a fenestrated endothelium, and thus hepatocytes can be relatively easily targeted with both viral and nonviral vectors; (3) despite the predominantly nonintegrative nature of AAV vectors, owing to the relatively slow turnover of mature hepatocytes, multiyear transgene expression after gene transfer to the liver has been documented in large animals and humans; (4) expression of a transgene in hepatocytes induces antigen-specific tolerance mediated by regulatory T-cells (Tregs); and (5) several preclinical studies demonstrate that it is possible to treat not only plasma protein deficiencies but also metabolic disorders with liver gene transfer, resulting in a long-term cure for many of these disorders in small and large animal models.

Liver gene transfer with AAV vectors has been tested in the clinic in the context of few indications, although the initial evidence of success obtained with liver gene transfer for hemophilia B paved the way for a number of clinical studies of AAV liver gene transfer ( Table 33.4 ). Initial results in the dog model of hemophilia B provided a strong rationale for targeting the liver to express the therapeutic FIX transgene. In the first AAV-FIX liver trial, a single-stranded AAV2 vector carrying the human FIX transgene expressed under the control of a liver-specific promoter was administered through the hepatic artery. This trial has been particularly important for the field of in vivo gene transfer because it demonstrated for the first time that it was possible to transduce the human liver with AAV vectors, leading to therapeutic levels of transgene expression. Additionally, it allowed the identification of important limitations of the approach related to vector immunogenicity and preexisting immunity to AAV in humans. Following the results obtained in the AAV2-FIX trial, a second trial was initiated in which a self-complementary AAV8 vector encoding for a codon-optimized version of the FIX transgene was administered intravenously to target the liver of hemophilia B subjects. In this study, a short course of immunosuppression was used to block potentially detrimental immune responses triggered by the viral vector, an approach also adopted in other GT trials. This initial trial successfully demonstrated that it was possible to target the liver via the administration of an AAV8 vector delivered through a peripheral vein. Additionally, it showed that transient immunosuppression could be safely applied with gene transfer to avoid detrimental immune responses, leading to long-term expression of the transgene product.

Table 33.4

Selected of Liver Gene Therapy Trials With AAV Vectors

Sponsor	Indication	AAV Serotype – Transgene	Stage (NCT Identifier)
University College London and Saint Jude Children’s Research Hospital	Hemophilia B	AAV8 – human FIX	Phase I/II (NCT00979238)
Spark Therapeutics and Pfizer	Hemophilia B	AAV-Spark100 – human FIX Padua	Phase I/II (NCT02484092; NCT03307980)
UniQure	Hemophilia B	AAV-5 – human FIX Padua	Phase III (NCT02396342)
Freeline Therapeutics	Hemophilia B	AAV-FLT180a – human FIX Padua	Phase I/II (NCT03369444)
Biomarin	Hemophilia A	AAV-5 – human factor VIII BDD	Phase III (NCT02576795; NCT03392974; NCT03370913)
Spark Therapeutics	Hemophilia A	AAV-Spark200 – human factor VIII BDD	Phase I/II (NCT03003533; NCT03432520)
University College London and Saint Jude Children’s Research Hospital	Hemophilia A	AAV-8 – human factor VIII v3	Phase I/II (NCT03001830)
Sangamo and Pfizer	Hemophilia A	AAV-5 – human factor VIII BDD	Phase I/II (NCT03061201)
Shire	Hemophilia A	AAV-8 – human factor VIII BDD	Phase I/II (NCT03370172)
Ultragenyx and Bayer	Hemophilia A	AAV-8 – human factor VIII BDD	Phase I/II (NCT03588299)
Audentes Therapeutics	Crigler-Najjar syndrome	AAV-8 – human UGT1A1	Phase I/II (NCT03223194)
Genethon	Crigler-Najjar syndrome	AAV-8 – human UGT1A1	Phase I/II (NCT03466463)
Adverum	Alpha 1 antitrypsin deficiency	AAV-Rh10 – human AAT	Phase I/II (NCT02168686)
Federico II University	Mucopolysaccharidosis type I	AAV-8 – human ARSB	Phase I/II (NCT03173521)
Ultragenyx	Ornitine transcarbamylase deficiency (OTC)	AAV-8 – human OTC	Phase I/II (NCT02991144)
Ultragenyx	Glycogen storage disease type 1a	AAV-8 – human G6PC	Phase I/II (NCT03517085)

ARSB , Arylsulfatase B; AAV, Adeno-associated virus; BDD, B domain-deleted; G6PC, glucose 6 phosphatase; UGT1A1, uridine diphosphoglucuronosyl transferase 1A1.

Delivery Tools (Vectors) With Advantages and Disadvantages for Each

Several gene delivery tools are available to transfer a transgene expression cassette to hepatocytes. These include both viral and nonviral vectors ( Table 33.5 ). The AAV vector platform is currently the most popular platform for liver gene transfer, although recent advances in manufacturing and encouraging results in animal models of hemophilia with lentiviral vectors have been documented. Owing to advances in the technology and potential benefits in terms of lack of preexisting immunity and theoretical ability to redose, nonviral delivery methods have recently gained attention as attractive tools for in vivo gene transfer to the liver. Both lentiviral vectors and nonviral delivery methods are currently being tested in animal models as tools for liver gene transfer for diseases like hemophilia and liver metabolic diseases, respectively.

Table 33.5

Main Liver Gene Delivery Tools Currently in Development, Pros and Cons

Vector	Pros	Cons
Nonviral vectors (based on nanoparticles)	Easy to manufacture; can accommodate large DNA fragments; no preexisting immunity; repeated dosing theoretically possible.	Lower efficiency compared with viral vectors; nuclear transport of genetic payload; persistence of transgene expression.
Lentiviral vectors	Low preexisting humoral immunity; persistence of transgene expression in dividing cells; induction of tolerance to the transgene.	Can transduce immune cells (dendritic cells, etc.); integrational mutagenesis; manufacturing can be challenging.
Adeno-associated virus vectors	Target the liver efficiently; can be manufactured in large amounts; do not integrate efficiently into the host genome; induction of tolerance to the transgene.	Preexisting immunity to the vector capsid; transgene expression does not persist in growing liver; vector readministration is challenging.

Advantages and Disadvantages of Different Gene Therapy Approaches (Gene Repair, Gene Addition, Gene Editing, Homologous Recombination, Nucleases, Promotorless)

For interpretation of clinical and preclinical studies as well as for discussing GT as a curative treatment for an LBMD, it is important to evaluate GT disease specifically and to differentiate between the multiple options of GT that have been described so far ( Figure 33.1 ). For this, it is also important to be aware of the stage of transition from preclinical to clinical applications for different methods. Next, we describe the methods that are already used in clinical studies or closest to this or that are the most important/innovative. Figure 33.1 gives an overview for this.

Fig. 33.1, Combination of different gene therapy approaches. As described in the text, a gene therapy (GT) approach is a combination of different aspects. (1) GT can be realized in vivo or in vitro , and (2) different possibilities for application of template DNA are possible, (3) the architecture of the template DNA can be different, and (4) GT can be assisted by nucleases. Further different ways of administration can be chosen. Different combinations of these aspects are possible and result in four main groups: GT with random integration, episomal GT, GT with targeted integration, and gene silencing.

First, in vivo and ex vivo GT must be differentiated. For clinical purposes, especially for LBMDs, ex vivo GT is less close to clinical transition.

Second, which vector (e.g., AAV) or kind of application (e.g., naked DNA, nanoparticle, exosome) is used (see also Table 33.5 ) has to be differentiated. For gene addition, the vector can also determine if the transmitted cDNA is integrated or not. A very recent study described a new option for GT with naked DNA and hydrodynamic injection, which was so far not suitable for clinical translation. But Khumbari et al. described a hydrodynamic injection in the biliary system via endoscopic retrograde cholangiopancreatography, which could overcome the problems hydrodynamic injection into blood would cause in humans.

Third, the architecture of the transmitted genetic information can be different, for example, a whole gene with or without flanking homologous arms for a specific area on the host genome or just parts of a gene for gene repair; furthermore, different promotor or enhancing sequences or codon optimizations can be added.

Fourth, there is the possibility to add tools to the introduced genetic information, such as nucleases (e.g., clustered regularly interspaced short palindromic repeat [CRISPR]/CRISPR-associated protein 9 [Cas9]) to facilitate targeted integration.

Different combinations from these four aspects are possible and also depend on the kind of GT that is intended. This can be gene repair, gene silencing, gene addition, or RNA splicing manipulation approaches. Table 33.6 provides a short overview of the different approaches and their characteristics, and the corresponding Figures 33.2–33.5 describe the approaches graphically.

Table 33.6

Gene Therapy Approaches and Their Characteristics

Approach	Description	Pros	Cons	Development stage
Gene addition -Episomal ( Fig. 33.2 ) -or random %integration -or targeted integration (HDR ± nucleases, e.g., Cas9) ( Fig. 33.3 ) - or Promoterless/ GeneRide strategy ( Fig. 33.4 )	Classic rAAV GT, easy to apply most expertise, can be enhanced by new techniques Retrovirus GT, lentivrus GT rAAV GT with homologous sequences added to the template DNA, integration via HDR at safe harbor locus (e.g., ROSA 26), can be enhanced with DSB caused by nucleases A new approach that uses the idea of targeted integration but instead of safe harbor locus, an organ-specific gene with high expression rate is targeted, so that the template DNA must not have a promotor. This approach can be performed with or without nucleases	Low risk of insertional mutagenesis or off-target effects Very stable transgene expression Very stable transgene expression also in the growing liver of young children, very low insertional mutagenesis, low off-target/random integration events (especially without nucleases) Very organ specific, use of the natural promotor, low insertional mutagenesis	Loss of template DNA/transgene expression because of cell turnover in young children (< 10 years) Risk of insertional mutagenesis Low efficiency (for no-nuclease approach); for nuclease approach, two vectors have to be applied and risk of off-target DSB Risk of disruption or mutation of targeted gene in case of nuclease application, low efficiency in case of no nuclease application	Clinical studies are done and running (CureCN), most translational approach at the moment, possibilities for readministration are under evaluation Nathwani et al. Meliani et al. not suitable for translation at the moment Rittelmeyer et al. very attractive approach and well working in animal studies, but there are still safety concerns for translation Junge et al. Landau et al. Very new and attractive approach but still far from translation Porro et al. Sharma et al.
Gene editing/repair HDR ± nucleases ( Fig. 33.5 )	In case of a small or point mutation, this part can be replaced by a correct template sequence with homologous arms. Again, efficiency without nucleases is low.	Very low risk for mutagenesis, using natural promotor for the specific gene.	In case of NHEJ instead of HDR events, a reserved activity of the mutated gene can be erased so that disease phenotype is declined. Only applicable for diseases with frequent mutation. Nucleases have to be designed for each disease or even different mutations.	Only applicable for a portion of diseases. Yang et al. ¹⁶⁶
*Ex vivo*	Hepatocytes, isolated by partial hepatectomy in the patient, are treated in vitro with gene therapy vectors; for this, all abovementioned approaches are possible.	Hepatocytes can be targeted directly, no side effects in other organs, lower vector doses are necessary.	Allogenic hepatocytes no option, autologous hepatocytes need a partial hepatectomy in a sick or small or liver-diseased patient.	Still focused on animal studies, extraction of hepatocytes is a problem VanLith et al.

Approach

Description

Pros

Cons

Development stage

Gene addition -Episomal

( Fig. 33.2 )

-or random %integration

-or targeted integration (HDR ± nucleases, e.g., Cas9)

( Fig. 33.3 )

- or Promoterless/
GeneRide strategy

( Fig. 33.4 )

Classic rAAV GT, easy to apply most expertise, can be enhanced by new techniques

Retrovirus GT, lentivrus GT

rAAV GT with homologous sequences added to the template DNA, integration via HDR at safe harbor locus (e.g., ROSA 26), can be enhanced with DSB caused by nucleases

A new approach that uses the idea of targeted integration but instead of safe harbor locus, an organ-specific gene with high expression rate is targeted, so that the template DNA must not have a promotor. This approach can be performed with or without nucleases

Low risk of insertional mutagenesis or off-target effects

Very stable transgene expression

Very stable transgene expression also in the growing liver of young children, very low insertional mutagenesis, low off-target/random integration events (especially without nucleases)
Very organ specific, use of the natural promotor, low insertional mutagenesis

Loss of template DNA/transgene expression because of cell turnover in young children (< 10 years)

Risk of insertional mutagenesis

Low efficiency (for no-nuclease approach); for nuclease approach, two vectors have to be applied and risk of off-target DSB

Risk of disruption or mutation of targeted gene in case of nuclease application, low efficiency in case of no nuclease application

Clinical studies are done and running (CureCN), most translational approach at the moment, possibilities for readministration are under evaluation
Nathwani et al.
Meliani et al.
not suitable for translation at the moment
Rittelmeyer et al.
very attractive approach and well working in animal studies, but there are still safety concerns for translation
Junge et al.
Landau et al.

Very new and attractive approach but still far from translation
Porro et al.
Sharma et al.

Gene editing/repair
HDR ± nucleases

( Fig. 33.5 )

In case of a small or point mutation, this part can be replaced by a correct template sequence with homologous arms. Again, efficiency without nucleases is low.

Very low risk for mutagenesis, using natural promotor for the specific gene.

In case of NHEJ instead of HDR events, a reserved activity of the mutated gene can be erased so that disease phenotype is declined. Only applicable for diseases with frequent mutation. Nucleases have to be designed for each disease or even different mutations.

Only applicable for a portion of diseases.
Yang et al. ¹⁶⁶

Ex vivo

Hepatocytes, isolated by partial hepatectomy in the patient, are treated in vitro with gene therapy vectors; for this, all abovementioned approaches are possible.

Hepatocytes can be targeted directly, no side effects in other organs, lower vector doses are necessary.

Allogenic hepatocytes no option, autologous hepatocytes need a partial hepatectomy in a sick or small or liver-diseased patient.

Still focused on animal studies, extraction of hepatocytes is a problem
VanLith et al.

DSB, Double-strand break; GT, gene therapy; HDR, homology-directed repair/homologous recombination, NHEJ, nonhomologous end joining; rAAV, recombinant adeno-associated virus.

Fig. 33.2, Episomal gene therapy. The classic recombinant adeno-associated virus (rAAV) gene therapy transports a template DNA with primer by endocytosis into the cell and following into the nucleus, where the template DNA stays episomal (in case of single-stranded template DNA after conversion to double-stranded DNA), and mRNA will be produced from this episomal concatemer.

Fig. 33.3, Homology-directed recombination is the biological mechanism that is used for targeted integration gene therapy. Without nucleases, the approach has a low efficiency because homologous recombination occurs without an induced double-strand break (DSB) only once in 1 × 10 5 cells. After induced DSB with nucleases, homology-directed repair/homologous recombination ( HDR ) occurs 100 to 10,000 times more frequently. Owing to the position between homologous arms, the template DNA (primer and transgene cDNA) will be included in the host genome, and the second strand will be produced complementarily. HA-L, Homologous arm left; HA-R, homologous arm right; ITR, inverted terminal repeats; PR, primer.

Fig. 33.4, Promoterless integration is a special approach of targeted integration gene therapy. The approach is similar to homology-directed repair/homologous recombination (HDR), but one homologous arm is a homolog to the human albumin locus and connected to the transgene cDNA with a 2A peptide (P2A) sequence but without its own promotor for the transgene cDNA. The second homologous arm includes the stop codon and homolog sequences to the area following the human albumin locus. Site-specific homologous recombination results in a fused mRNA (albumin-P2A-transgene cDNA) transcribed by the strong albumin promoter. Because of the P2A, it is translated into two separate proteins. GOI, Gene of interest; HA, homologous arm; P2A, 2A peptide.

Fig. 33.5, Gene repair approaches repair particular short-mutated regions of genes by homologous recombination. For example, the wild-type gene has guanine at position 4 and the mutated gene in the patient an adenine. This is repaired by homologous recombination with the wild-type sequence, which was delivered to the cells by single-stranded rAAV. A, Adenine; G, guanine; rAAV (ss), recombinant adeno-associated virus (single stranded).

Disease-Specific Aspects and Milestones

Next, we will describe exemplary some disease-specific aspects and suitability for GT as well as important disease-specific study results. Fig. 33.6 gives an overview of disease-specific suitability for GT.

Fig. 33.6, Overview characteristics of diseases with regard to gene therapy applicability.

Crigler-Najjar Syndrome

Crigler-Najjar syndrome (CNS) is a recessive inherited disorder caused by a deficiency of uridine diphosphoglucuronosyl transferase 1A1 (UGT1A1). Because of missing hepatic bilirubin glucuronidation, patients experience accumulation of unconjugated bilirubin in serum, leading to a risk of irreversible damage to the central nervous system. For CNS, a natural mutant rat model exists, the Gunn rat, and also a transgenic mouse model was described. The mouse model requires phototherapy for survival to adulthood, whether or not it’s the Gunn rat. The CNS is an ideal model for GT because liver parenchyma in classical CNS is considered structurally and histologically normal; however, it is reported that over time, some fibrosis can develop. Further, the correction level needed for sufficient phenotypic correction is low—only 5% of normal UGT1A1 activity. Finally, the success of the intervention is easily monitored by serum bilirubin levels. Up to now, the only available conservative therapy to individuals affected by the severe form of CNS is phototherapy, which has major implications on the quality of life and loses efficacy during adolescence. LT during adolescence is often considered to be the only curative option available to prevent acute decompensation and irreversible brain damage. For CNS, various GT approaches have been tested in animal models. The rAAV approach seems to be the most promising. Seppen et al. showed a 70% reduction of serum bilirubin levels following AAV gene transfer for UGT1A1. The dose used in this study (2.5–5 × 10 ¹² vector genomes [vg]/kg body weight) was similar to that used in the high-dose cohort of the rAAV8 trial of GT for hemophilia B, which showed efficacy, although with detection of immune responses against the viral capsid in some patients. In the UGT1A1 knockout mouse model, Bortolussi et al. were able to show phenotypic correction after intraperitoneal injection of AAV9 UGT1A1 vectors in neonate mice.

More recent work in Gunn rats shows that AAV8-mediated gene transfer to the liver using an optimized UGT1A1 expression cassette results in correction of bilirubin levels at doses lower than those tested so far, and also, studies in the mouse model showed sufficient therapy with 2.5 × 10 ¹¹ vg/kg body weight.

Like in many LBMDs, it would be suitable to treat the disease in children, first, to eliminate the risk for neurological damages, second, to avoid the quality of life impairing phototherapy as early as possible, and third, to avoid development of liver fibrosis (based on very recent findings ). But this is difficult because of the described problem of transgene-loss in rAAV8 episomal GT in the growing liver.

Two recent studies tested the readministration in the Crigler-Najjar mouse model with success. Greig et al. readministered the same vector on day 56 after first injection within the first 24 hours of life. Because of the very early first injection, mice did not develop antibodies, which is also described by Wang et al. However, this approach is very unlikely to be transferred to the clinic because such an early diagnosis in the newborn is impossible except when genetic testing is performed in pregnancy because of known risk. More translational is the approach of Bockor et al., with serotype switching for readministration. For this approach, the need for the approval of two products could hamper clinical translation. Better solutions could be to find possibilities for readministration or targeted integration (see “AAV Persistence in the Developing Liver”). One very attractive targeted integration approach was studied in the Crigler-Najjar mouse model by Porro and colleagues. They could reduce bilirubin levels in the mice by intraperitoneal injection of rAAV2/8 packed with a template DNA consisting of homologous arms for the albumin locus so that UGT1A1 cDNA is integrated into the host genome at the end of the albumin reading frame separated from the albumin by a 2a peptide. This approach reached 5% enzyme activity of the wild type, enough for significant reduction of serum bilirubin levels, without any nuclease-enhancing homology-directed repair/homologous recombination (HDR) but with high vector doses (1 × 10 ¹² vg/mouse). Therefore for clinical translation, this approach needs to be optimized.

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