# Presentation: 880872

## SAFETY FEATURES IN THE DESIGN, 

**SAFETY FEATURES IN THE DESIGN, **
**MANUFACTURE  AND CLINICAL MONITORING **
**OF LENTIVECTORS FOR THE TREATMENT OF **
**PARKINSON’S DISEASE, PROSTATE **
**CANCER AND AIDS.**

**FDA/BRMA 25-26 Oct 2001**
**Susan M Kingsman**
**Senior Vice President**
**Research**

**Notes:**
Oxford BioMedica is a gene technology company that develops viral based gene transfer systems for therapeutic and discovery purposes. We are very pleased to have this opportunity to present our work on LentiVectors and to contribute to the development of the regulatory framework that will underpin the translation of these vectors to the clinic for their evaluation for the treatment of a range of diseases. We are currently engaged in a phase I/II trial of a retroviral vector in breast and ovarian cancer and we propose that similar manufacturing and safety testing standards can be applied to LentiVectors.

## The Retroviridae

**Spumavirus**

**Mammalian**
**C-type**

**Lentivirus**

**D-type**

**B-type**

**Avian**
**C-type**

**BLV/**
**HTLV**

**Most Vectors**

**All share RT(*****pol*****) but differ in morphology, pathogenicity**
**and replication and gene expression characteristics**

**Notes:**
Lentiviruses are in the Retroviridae.  We can therefore use concepts established for type C viruses that have already been used in the clinic.  However the route to implementing these concepts for Lentiviruses has not always been obvious and easy. Some aspects of LentiVector design and assay will therefore be familiar to you and others will be novel.

One important point is that, unlike C-type retroviruses, the lentiviruses have not been associated with cancer despite producing very high viraemic states over extended periods. 

## Lentiviruses: Analysis of Pol Sequences

**Human Immunodeficiency Virus, HIV: A primate lentivirus**

**Lentiviruses: Analysis of Pol Sequences**

**Primate**
**Lentiviruses**

**Non-Primate**
**Lentiviruses**

**Notes:**
Lentiviruses are diverse with little primary nucleic acid sequence homology but the Pol coding sequence can be related. Non-primate lentiviruses are very divergent from primate lentiviruses but share features of genetic organization. 

We have chosen to study HIV-1 as the archetypal lentivirus and EIAV as an example of a non-primate, non-immunodeficiency virus. Most of this talk will be confined to EIAV.

## Why Develop Lentivirus-Based Vectors?

**Unique Advantages for Gene Therapy**

** ****Vectors for long term, stable therapy of chronic diseases**
** ****Vectors for unmet medical needs**

**1. Simplicity**
**2. Up to 11kb capacity**
**3. Defined integration of genes**
**4. Long term expression**
**5. Transduction of non-dividing cells**
**6. Transduction of slowly dividing cells**

**Notes:**
There are very good reasons to develop these viruses as vectors and the next few slides will give a lightening overview of some of the attributes and applications of LentiVectors.

## EIAV Vectors: Gene Transfer to the Nervous System

**Hippocampus/septal cholinergic system:**
**Site of learning & memory **
**Major damage in ****stroke**
**Primary target in ****Alzheimer’s**

**Substantia nigra:**
**Primary target in **
**Parkinson’s**

**Striatum:**
**Primary target in **
**Huntington’s**

**Nucleus acumbens:**
**site of ****addictions**

**Spinal motoneurons:**
**Primary target in**
**ALS, SMN**

**Muscle**

**EIAV Vectors: Gene Transfer to the Nervous System**

**A powerful approach to the management of neurological  disease**

**Rat **

**Notes:**
EIAV can transfer genes to many different neuronal types. When pseudotyped with the VSV-G envelope  the expression is localised. When pseudotyped with the rabies G envelope expression is distributed by retrograde transport along axons to connecting cell bodies. This allows peripheral administration to the CNS. The data shown have been obtained in the rat.

## EIAV Vectors: Long Term Gene 

**Expression *****in Vivo***

**8 days**

**24 weeks**

**Sustained expression minimises the need for repeat invasive delivery**

**Rat thalamus**

**Notes:**
In our observations to date, EIAV vectors are not shut down intrinsically or extrinsically. This is a significant difference from MLV vectors where genetic silencing often occurs and from some other vectors such as herpes or adenovirus based systems where there are significant immunological responses.

## EIAV Vectors: No Systemic Toxicity at 

**80 rats**
**14d, no clinical signs of toxicity **

**Intra-cranial (10****7****)**
**Over 100 rats**
**Up to 8mths**
**No clinical signs**
**No histological abnormality**
**Mild acute inflammation **

**EIAV Vectors: No Systemic Toxicity at **
**Doses Relevant to the Clinic**

**Tail vein (10****7****)**
**12 rats 21d**
**No liver tox or damage by histology**
**No vector in liver or lung**

**Following MLV toxicity and biodistribution protocols**

** ****Intra-tumoral (10****7)**** **
** ****21d No abnormal tox **

**Notes:**
Toxicity and bio-distribution are being studied in the rodent models for the specific target diseases. Similar protocols and endpoints to those used to support our current retroviral (MetXia) vector clinical trial are being followed. To date we observed no toxicity but this is an ongoing study to generate a sound pre-clinical data base.

## An EIAV-Based Vector for The 

**Tonic expression of dopamine, augmentation of L-DOPA therapy**

**An EIAV-Based Vector for The **
**Treatment of Parkinson’s Disease**

***IRES***

**SIN ****LTR**

**TH**

**CH1**

**AADC**

**CMVp**

***IRES***

**Aromatic AA**
**Dopa Decarboxylase **
**(AADC)**

- 6-OHDA lesion

- Control

**Notes:**
We are actively developing products based on LentiVectors.  This product is an in situ biochemical factory created by delivering three key enzymes encoded in a single polycistronic LentiVector by stereotactic injection into the striatum.  This provides tonic levels of dopamine and also provides AADC to metabolise L-Dopa given in conventional therapy.  

Efficacy is being established in relevant animal models. 

## An EIAV Based Vector for 

**An EIAV Based Vector for **
**the Treatment of Late Stage Prostate Cancer**

***Slow growing tumour ***
***100 fold improved TK***
***TK clinical experience***
***Prodrug strategy terminates therapy easily***
***Therapy selective for dividing cells***
***Recurrent intractable malignant disease***
***Local delivery with transrectal ultrasound guidance***

**MCA Preliminary discussions on LentiVectors**
**GTAC and MCA protocol outline early 2002**

**LNCaP**

**Efficacy in progress**

**PC-3 xenograft**

**Abs**

**0**

**20**

**40**

**60**

**80**

**100**

**120**

**% Control**

**GFP**

**-**

**TKm**

**Wst-1 assay/PC3**

**PURO**

**CMVp**

**TKm**

**CMVp**

**Notes:**
This product uses  a conservative GDEPT strategy for the treatment of prostate cancer. It uses promoters and the TK therapeutic gene that have already been in the clinic. The phase I/II trail will test the efficiency of gene transfer to this very slowly dividing cell population and the potency of an improved TK gene. The patient population have no other treatment options. We intend to present a protocol and product for consideration by GTAC and the MCA early next year.

## LentiVector Specification

**LentiVector Specification**

***Basic Design***
***and Production***

***Enhanced Design***
***and Production***

***Gene specific considerations***

***Disease and patient specific considerations***

**Growth factors**

**Prodrug activating enzymes**

**Metabolic replacement**

**Cell death regulators**

**Terminal disease**

**Chronic disease**

**Adults**

**Children**

**Specification is an on-going process. Clinical evaluation should occur**
**in parallel with vector and process development for certain indications**

**Notes:**
Our approach to LentiVector development is to translate to the clinic as early as possible while conducting continued R and D in parallel. Many questions can only be answered in the relevant host i.e. man and there are certain gene and disease scenarios that lend themselves to ethical early testing.  

## LentiVectors : 

**Key Vector-Specific Safety Issues**

**Replication Competent Lentiviruses (RCL) **
**in the product**
*** ******Minimise the generation and impact of RCLs***
**Mobilisation of the transfer vector in target cells**
***Minimise the inappropriate dissemination of the transgene***

**Notes:**
There are two key vector-specific safety issues. I will focus on these and show how we have been developing vectors to address these issues. 

## Minimising the Generation 

**and Impact of RCLs**

**If possible use a non-pathogenic virus**
**Split the vector production system into at least three components**
**If possible use a stable producer cell line**
**Eliminate all non-essential coding and *****cis*****-active sequences**
**Minimise the potential for homologous recombination**
**Reduce packaging of vector helper components**
**Use sensitive calibrated assays for detecting RCLs**

**Notes:**
We have identified six approaches for minimising the generation and impact of RCLs. I will discuss each of these. In addition it is of course not enough to design away RCLs we have to validate this by testing and I will describe our RCL assay procedure. Much of what I will discuss is work in progress and will continue to be refined and supplemented. However all of the major conclusions are supportable and we will be publishing our protocols and data.

## Minimising the Generation 

**Consequences of any RCL may be minimised**

**Minimising the Generation **
**and Impact of RCL**

## Features of HIV and EIAV 

**Features of HIV and EIAV **

**HIV**

** ****Extensive analysis **
** ****Complex (6 accessory genes)**
** ****Human pathogen**
** ****Replicates in human cells**
** ****Fatal immunodeficiency**
** ****Global pandemic**
** ****30M AIDS/HIV+ve (worldwide)**

**EIAV**

** ****Moderate analysis**
** ****Simple (3 accessory genes)**
** ****Equine pathogen**
** ****No replication in human cells**
** ****Self limiting anaemia**
** ****Endemic in horses in the Tropics**
** ****Rare disease in stables (worldwide) **

** ****Different lentiviruses have different safety profiles**
** ****EIAV is rarely fatal in horses, does not cause immunodeficiency**
** ****No *****a priori***** expectation that EIAV would be a human pathogen**
** ****Low probability of a patient encountering EIAV **

**Notes:**
The attraction of HIV is the level of detailed knowledge that is available although research into the non-primate lentiviruses is now catching up. HIV causes disease in man although we still do not know all of the mechanistic details.  EIAV does not usually cause a fatal disease in horses and  does not cause immunodeficiency and so there is no reason to think that any vector-derived RCL could acquire these characteristics.  The chance of a patient encountering EIAV is slim and so mobilisation and genetic interactions between an EIAV vector and virus are highly unlikely.  

If there is no potency and efficacy difference between HIV and EIAV for a particular therapy we favour using EIAV. It is however too soon to determine whether one specific lentivirus will be universally useful as a vector system.

## Minimising the Generation 

**Split the vector production system**
**into at least three components**

**Increases the number of recombination events required to generate an RCL**

**Minimising the Generation **
**and Impact of RCL**

## LentiVectors: The Basic System

**LentiVectors: The Basic System**

**gag-pol**

**env**

**RNA**

**1**

**2**

**3**

**3 Component**
**Split System**

***Vector genome***

**Therapeutic Gene(s)**

**Low probability of generating RCLs requiring four cross overs, two non-homologous**

**2**

**3**

**1**

**Notes:**
The  basic safety feature for any retroviral or lentiviral vector system is to split the components into at least three entities. The transfer vector, the Gag-Pol expression plasmid and the envelope expression plasmid.  The latter two being the packaging or helper plasmids. This strategy has been proven to be vastly superior for the safe production of retroviral vectors and there appears to be no good reason not to adopt this approach with LentiVectors. 

## Minimising the Generation 

**Minimising the Generation **
**and Impact of RCLs**

** ****DNA recombination unlikely**
** ****Genetically stable**
** ****Conventional  manufacturing parameters established**
** ****Characterised starting materials**

**Notes:**
Stable cell lines are usually considered preferable for product manufacture because of ease of scale, stability and historical experience with retroviral vectors. They have the advantage that DNA recombination between vector components is highly unlikely. 
It is possible however that there may be operational reasons for using a transient system, for example some envelopes  and Gag-Pols are cytotoxic when continually expressed and may be best used in transient systems. It is certainly quicker to produce vector transiently than to select for a stable cell line and this may be an advantage to accelerate the early phases of clinical evaluation. Transient systems could be scaled and adapted to GMP conditions. The high plasmid copy numbers involved could however lead to recombination and so minimising the potential for this and using effective RCL testing will be critical. There may also be a case for developing mixed systems where one or more component is stably configured in a cell and the others are delivered transiently.
We would suggest that whether stable lines or transient systems are used the manufacturing process should be adequately designed to guard against RCLs and also be shown to be free from RCLs and undertaken as a GMP process. 

## Minimising the Generation 

***cis*****-active sequences**

**Reduces recombination and pathogenic potential**

**Minimising the Generation **
**and Impact of RCL**

## Lentiviral ProViral Genomes

**Transfer vector construction is complicated due to multiple introns and accessory genes**

***EIAV: Non-Primate, 3 accessory genes***

**SD**

**LTR**

**gag**

**pol**

**S2**

**env**

**rev**

**rev**

**tat**

**LTR**

**RT  RH ****  **** ****   ****DU      IN**

**tat**

****

**cPPT**

**RRE**

**Pu****n**

**TAR**

**RRE**

***HIV: Primate, 6 accessory genes***

**SD**

**vpr**

**vpu**

**vif**

**pol**

**RT****      ****  RH****          **** IN**

**nef**

**tat**

**rev**

**env**

****

**Pu****n**

**TAR**

**cPPT**

**Notes:**
This busy slide serves to show that although the aim of removing unnecessary sequences is a good one it is not necessarily obvious how to achieve the aim. This is because these viruses are loaded with introns, cis active sequences that are critical for replication and gene expression and multiple additional genes whose function is or was unknown.

This is why LentiVector development from the first suggestion in the early 1990s that HIV might be a vector, to date, has appeared to be slow as the next incremental improvement is only gradually achieved to result in a new generation of vectors. 

## The ‘Additional’ Genes of Lentiviruses

**+**** = Tat, Nef, Vif, Vpu, Vpr, potential pathogenicity factors in man**

**Notes:**
Why should we bother to remove additional genes. Simple, we do not know how Lentiviruses cause disease and we do not know the full biochemical and pathogenic potential of these additional genes. If the genes encode structural or essential proteins then we have no option but to retain them but if they are truly accessory then they should be jettisoned. For all lentiviruses the only accessory gene for which no overt pathogenic property has been ascribed is Rev.

Reviewed for HIV in, 
S.Bour and K. Strebel (2000). HIV accessory proteins: Multifunctional components of a complex system. Adv Pharmacol. 48, 75.

Reviewed for EIAV in 
Yoon S, Kingsman SM, Kingsman AJ, Wilson SA, Mitrophanous KA., Characterization of the equine infectious anaemia virus S2 protein, J Gen Virol 2000 Sep;81 Pt 9:2189-94; 

## 1st Generation EIAV Vector Producer Cell:8Z-20

**Issues****: Recombination homology, S2 and Rev expressed**** in production**

***John Olsen’s***
***BiG-45***
***Packaging***
***Line***
*** ***

****

**Notes:**
Our first generation producer cell achieved some of the aims. Working with John Olsen at UNC we inactivated the Env, S2, Rev and Tat coding regions in the transfer vector and provided a Gag-Pol expression cassette that had all of the leader sequence upstream of the SD deleted to ablate the presumptive packaging site. 
However this configuration provides two significant regions for homologous recombination. It also causes Rev and S2 to be expressed in the packaging cell although we showed that S2 does not  appear in the vector preps.

(Yoon S, Kingsman SM, Kingsman AJ, Wilson SA, Mitrophanous KA., Characterization of the equine infectious anaemia virus S2 protein, J Gen Virol 2000 Sep;81 Pt 9:2189-94; )
(Yamada K, Olsen JC, Patel M, Rao KW, Walsh CE; Functional correction of Fanconi anemia group C hematopoietic cells by the use of a novel lentiviral vector; Mol Ther 2001 Apr;3(4):485-90)
(Olsen JC; Gene transfer vectors derived from equine infectious anemia virus; Gene Ther 1998 Nov;5(11):1481-7)

The producer cell line 8Z-20 is proving very useful.

## VIRUS YIELD FROM 8Z-20 PRODUCER

** **

** **

- 1x10

- 6

**Time (hours)**

** **

- 1x10

- 5

- 1x10

- 4

- 1x10

- 3

**Viral Titer **

**(TU/ml)**

**0**

**-**

**24**

** **

**24**

**-**

**48**

** **

**48**

**-**

**72**

** **

**96**

**-**

**120**

** **

**72**

**-**

**96**

** **

**Sodium butyrate**

** **

**Doxycycline**

** **

- +

- -

- -

- -

- -

- +

- +

- +

- +

- +

**Stable producer line constructed**
**Scaled in roller bottles >10****6**** t.d.u./ml for 5 days**
**Standard vector for benchmarking improvements**

**Notes:**
We now have a consistent supply of a basic LentiVector that can be used as cross reference between studies. We can now benchmark improvements against this vector performance. It is, for example, of little use making a vector that is perceived to be safer but which can only achieve a production titre of 100 particles per ml. Safety, utility and practicality must be quantified and balanced. The parameters of RCL assays can be evaluated at scale with this cell line

## EIAV Minimal Transfer Vector

** ****Minimal transfer ****vectors do not express any EIAV**** proteins****.**** **
** ****>8kb insert capacity.  **
**  ****Homology at ******
** ****cPPT and RRE optimise yields or potency but create regions of  homology**

***pONY8.1 ******series: GFP, LacZ, (optional cPPT, WPRE, RRE)***

***pEV53B***

**RRE**

****

**pA**

**Rev**

**Rev**

**Pol**

**S2**

**CMVp**

**SD**

*****

**Gag**

**cPPT**

**Notes:**
Our next step was to improve the transfer vector
The EIAV transfer vector genome can be stripped down to leave about 1100 nucleotides of EIAV sequence that contains only the essential cis active regions for packaging ) reverse transcription (R, Pun and in some cells cPPT)

The packaging plasmid for the expression of Gag-Pol in our BiG-45 packaging cells was the next issue to address. It contains part of the packaging site in the amino terminal region of Gag, it contains RRE because, as with HIV, the Rev/RRE interaction is essential for Gag-Pol expression, it contains the cPPT as part of Pol. These regions provide potential sites for homologous recombination.

## Minimising the Generation 

**Minimise the potential for homologous recombination**

**Remove all homologous sequences from the Gag-Pol packaging plasmid**
**Do not compromise expression of Gag-Pol**
**Examine requirement for RRE/Rev**

**Minimising the Generation **
**and Impact of RCL**

***pEV53B***

****

**pA**

**Rev**

**Rev**

**Pol**

**S2**

**CMVp**

**SD**

*****

**Gag**

**cPPT**

**RRE**

**Notes:**
This is not as easy as it sounds. The plasmid must continue to encode Gag-Pol so we cannot make significant changes such as deletion and the RRE element is large and is essential for Gag-Pol expression.

## Lentivirus Codon Usage

**Lentiviruses maintain a suboptimal codon bias**

**Notes:**
The Gag-Pol sequence can be altered by making use of the redundancy of the genetic code. This would not be a good strategy however if such changes in codons altered the efficiency of expression. Lentiviruses are unusual in maintaining an appalling codon bias as far as the tRNA bias in mammalian cells is concerned. We decided to change both the Gag and Pol sequence so that the packaging site and the cPPT were changed and we modified the majority of the codons in the hope that expression would be improved by optimising the codon usage. This was done first with HIV and subsequently with EIAV.

We discovered that expression of the codon-optimised Gag-Pol was good and in the case of HIV it was improved. A particular bonus however was that in both cases expression was independent of Rev. The provision of Rev made no difference to the kinetics or levels of Gag-Pol expression or to the vector titres that could be obtained.

## Codon Optimised Gag-Pol

** ****Codons changed across *****gag-pol***** except in the critical frame-shifting overlap region**
** ****Removes all blocks of sequence homology**
** ****Rev-independent**
** ****Achieved for EIAV and HIV *****gag-pol***

***pESYNGP***

***pEV53B***

****

**RRE**

**pA**

**Rev**

**Rev**

**Pol**

**S2**

**CMVp**

**SD**

*****

**Gag**

**cPPT**

**Notes:**
The Rev-independence allowed us to jettison all of the RRE region. The synthetic Gag-Pol gene contains no authentic EIAV sequences flanking the Gag-Pol coding sequence. The coding sequence has been changed except for a short sequence that spans the critical frameshifting overlap.

## Minimal EIAV Vector System

** ****No obligate requirement for **_**any**_** accessory genes but Rev/RRE can improve yields**
** ****No functional viral proteins or significant coding regions in the transfer vector**
** ****Transfer vector contains only 1100 nt of original EIAV nucleic acid**
** ****No significant ****homology between components**
** ****Approved in UK by HSE for CL1 use with non-toxic genes  i.e. lowest containment**
** ****Similar HIV-1 system has been constructed at OBM**

***pTOG***

***pESYNGP***

**cPPT**

***pONY8.1***

**Notes:**
We now have a minimal vector system. The transfer vector has no regions of homology with the packaging vectors. We have found that under some circumstances the addition of RRE to the transfer vector and the provision of  Rev increases the production yield. Also in some cell types the addition of the cPPT enhances gene transfer. If transfer vectors need to be configured with these additional cis-active sequences there is still no homology with the synthetic gag-pol gene. 

It should be noted that the Rev protein has not been associated with any overt pathogenic properties of any lentivirus and we propose that expression in the packaging system could therefore be acceptable

## 2nd Generation Packaging Cell:

**ESYN-29-iG Series**

***pTOG + pcDNA6/TR***

**VSV-G**

**TetO****2**

**CMVp**

**pA**

***TetR***

**CMVp**

**pA**

***pTO-Rev ***

**Rev**

**TetO****2**

**CMVp**

**pA**

**Optional codon optimised Rev**

***pESYNGP***

**Work in progress in collaboration with John Olsen: **
**The system aims to meet the 8Z-20 production characteristics **

**Notes:**
A second generation packaging line is now being characterised and a new benchmark producer is being generated. As before we use the Tet-on system to regulate VSV-G as this is toxic to cells in high levels. The EIAV SYNGP is not toxic and is made constitutively.

Rev is an option because occasionally titres are improved via an effect on the transfer vector.  This has been codon-optimised for efficiency.

We have yet to establish the performance characteristics of this second generation packaging line. To date yields from transient production systems have been good and we are optimistic that this will be carried through into stable production. 

The minimal vectors may also be suitable for scaled transient production because of the zero potential for homologous recombination.

## Minimising the Generation 

**Reduce packaging of vector helper components**

**Lessons from retroviral vectors: Eliminate packaging and dimerisation signals**

**Minimising the Generation **
**and Impact of RCL**

**Notes:**
Work with Retroviral vectors showed that RCRs were generated during virion production via RNA: RNA interactions in the particle These interactions could be homologous or illegitimate i.e non-sequence specific. The latter are far less frequent but RT can readily switch templates and so once an RNA is co-packaged it can be a substrate for illegitimate recombination. Having excluded homologous recombination we then wished to determine the likelihood of  co-packaging of vector components that might lead to illegitimate recombination.

## Self Packaging Gag-Pol Transcripts

**XX**

**Self Packaging Gag-Pol Transcripts**

**Gag-Pol**

- CMV

- CMV

**STOP**

**Gag**

- CMV

**WT ******

**Gag-Pol **
**expression plasmids**

** ******** ****(various)******

** ********(Synthetic)******

**RNA only control**

****

**U3**

**R**

**U5**

**Gag-Pol**

**PBS**

** ****Defining sequences that allow Gag-Pol proteins to  package the cognate mRNA**
** ****Defining sequences that allow co-packaging of Gag-Pol and transfer vector RNA**

**Gag-Pol**

**Notes:**
Gag-Pol can package its cognate mRNA and in fact co-translational packaging is a key feature of some primate lentiviruses (e.g. HIV-2). To test for packaging of the helper Gag-Pol mRNA  we made complete and partial deletions in the presumptive packaging site. We measured the amount of the Gag-Pol RNA in the cell and in VLPs to obtain a measure of packaging. Quantitative PCR methods (TaqMan) were used and a range of controls for comparative gene expression were included. The study was done with either the Gag-Pol expression plasmid alone or in the presence of a wild type packageable transfer vector to mimic the situation of co-infection.

## Self-packaging of EIAV Gag-Pol mRNA

**Partial or complete removal of ********reduces packaging to background levels**
**No evidence for ‘piggy backing’ via dimerisation with the transfer vector**

**Notes:**
We have been able to develop a quantitative packaging and co-packaging assay.  The smaller the ratio of VLP associated RNA to cell associated RNA the lower the packaging efficiency

Neither the partial or completely ablated packaging site EIAV vectors managed to package their cognate RNA above background. Furthermore there was no evidence for dimerisation of the packaging Gag-Pol RNA with the vector RNA.

We cannot exclude some background level of packaging. However given the lack of homologous recombination, the relative inefficiency of illegitimate recombination and the low probability of a meaningful entity emerging from the requisite two illegitimate events we believe that we now have a vector system where the generation of RCLs is highly improbable.

## Minimising the Generation 

**and Impact of RCLs**

**The assays must relate to the properties of any RCL**

**Use sensitive, calibrated assays to detect RCLs**

**Notes:**
We have designed our LentiVectors such that the only route to generating RCLs is via illegitimate  recombination in the vector particle as a result of random background packaging.  We therefore believe that the generation of an RCL is improbable. We have now begun to develop relevant tests to confirm the absence of RCLs in the clinical product.

## Theoretical RCL structures:

**All RCL’s have  Gag-Pol**
**The most likely Gag-Pol is vector derived**
**Any other Gag-Pol is only rendered transmissible with**
**	****vector derived Env **

**Theoretical RCL structures:**
**A Generic View**

**Screen for transmissible reverse transcriptase (Pol).**
**Qualify ambiguous results by a second screen for trasmissible Gag and Pol and by screening for transmissible Env **

**Notes:**
To test for RCLs we considered the nature of the entities that we were seeking to detect. It is by definition however impossible to speculate on the nature of an RCL that could arise by illegitimate recombination either with vector components or with some unspecified element. Furthermore we did not wish to restrict our assays by the VSV-G envelope that is currently being used as this is likely to be different in different therapeutic vectors. We therefore sought to develop generic assays that would apply to any RCL. The defining feature of an RCL is the reverse transcriptase. The secondary feature is the RT in the context of Gag and Env sequences. All RCLs that arise as a consequence of the vector system will have one of the input components i.e. either Gag or Env.  We can therefore assay for RT and if any ambiguous results are obtained use a secondary assay to determine if there is also any transmissible Gag or Env.  If any non-vector derived elements contribute to RCLs the end result will still be a positive signal for RT and for either the EIAV Gag-pol or the Env.

## Proposed Testing for RCL

** ****Test vector preparations and post-production cells **
**   ****following current CBER guidelines for MLV derived vectors**
** ****Use F-PERT assay as the primary assay tool**
**   *****Fluorescence-based Product Enhanced Reverse Transcriptase Assay***
** ****Use PCR assay to resolve ambiguous PERT assay results**
**   *****Specific assay for hypothetical recombinant molecular structures***
**	**

**We have developed these assays and strategies in collaboration and discussion with relevant UK Government agencies (LGC, NIBSC) and with a contract manufacturer, Q-One Biotech **

**Notes:**
To assay for RCLs we propose to follow the CBER guidelines for MLV using appropriately modified assays to test 100ml of supernatant and 1% or 10e8 post-production cells. The assays will be capable of detecting a single RCL or RCL producing cell. Amplification/co-cultivation protocols will use the 14day period that has been shown to amplify a single RCR to allow a yes/no positivity read out. The assays are biochemical assays that are highly sensitive and reproducible and that can readily be standardised and transferred to the manufacturer.

## F-PERT: Product Enhanced Reverse 

**Transcriptase Assay**
**(MLV, AMV, HIV-1/2, EIAV, SRV-1, HTLV-1/2, FLV, FIV, BIV, CAEV, **
**MVV, PoERV, SFV, SMRV)**

**ABI Prism******** 7700**
**(TaqMan)**

**pol**
**(10-100/virion)**

**Disrupted VLP**

**Reverse transcription of MS2 RNA**

**NP-40**

**MS2 cDNA**

** ****Independent of the nature of events leading to RCLs **
** ****Broadly applicable and high sensitivity (10-100 ****partic****les)**
** ****Modified to control against  false positives **

**Notes:**
The PERT method is well established. In essence any RT in the prep will reverse transcribe the MS2  phage target RNA from a sequence specific primer. The resulting cDNA is then amplified and quantified by PCR with a specific primer pair using Taqman.

Silver J, Maudru T, Fujita K, Repaske R. An RT-PCR assay for the enzyme activity of reverse transcriptase capable of detecting single virions.  Nucleic Acids Res. 1993 Jul 25;21(15):3593-4

Boni J, Pyra H, Schupbach J Sensitive detection and quantification of particle-associated reverse transcriptase in plasma of HIV-1-infected individuals by the product-enhanced reverse transcriptase (PERT) assay.  J Med Virol. 1996 May;49(1):23-8

Lovatt A, Black J, Galbraith D, Doherty I, Moran MW, Shepherd AJ, Griffen A, Bailey A, Wilson N, Smith KT High throughput detection of retrovirus-associated reverse transcriptase using an improved fluorescent product enhanced reverse transcriptase assay and its comparison to conventional detection methods.  J Virol Methods. 1999 Oct;82(2):185-200.

## The F-PERT Assay Is Highly Sensitive

**10**

**12**

**14**

**16**

**18**

**20**

**22**

**24**

**26**

**28**

**30**

**32**

**34**

**36**

**38**

**40**

**42**

**MLV neat**

**MLV-1**

**MLV-2**

**MLV-3**

**MLV-4**

**MLV-5**

**EIAV neat**

**EIAV-1**

**EIAV-2**

**EIAV-3**

**EIAV-4**

**EIAV-5**

**MLV undisrupted**

**EIAV undisrupted**

**Media**

**Media/DB**

**water/DB**

**water**

**MS2cDNA**

**Ct**

**The F-PERT Assay Is Highly Sensitive**

**Both MLV (Mn****2+****-dependent) and EIAV (Mg****2+****-dependent) RT activities **
**were detected by PERT assay over a wide range of dilutions.**

**Notes:**
We have calibrated the F-PERT assay to ensure that different RTs can be detected at high sensitivity. Studies are ongoing to provide comparative data for various spiked supernatants.

## PCR Assay:  Nested Primers For The Detection Of

**PCR Assay:  Nested Primers For The Detection Of**
***gag*****, *****pol***** and *****VSV-G***

** ****Nested primers detect *****gag*****, *****pol***** and *****env*****, separately or as a linkage group**
** ****No cross reaction with other retroviruses or lentiviruses**
** ****Developed in collaboration with the Laboratory of the Government Chemist**

**Notes:**
We have devised a PCR test for Gag-Pol and Env. Our strategy has been to develop a set of nested primer pairs that can detect all of the packaging components either singly or linked. At present we have primers for VSV-G but we will develop sets for other envelopes.  

## Slide 38

**Notes:**
We have determined the detection level in various spiked mixtures and set a test protocol that must detect 1 to 10 copies of all packaging components in a background of 100,000 genomic copies. This test is then applied to an assay system that allows amplification of RCLs.

## RCL Amplification and Detection

**Transduction by test article**

**Split cells at appropriate ratio over appropriate time**

**PERT assay on cell-free supernatants**

**PCR assay for EIAV *****gag*****, *****pol*****  and *****env***** on genomic DNA**

**Amplification process is essential**
**Readily adapted for screening producer cells **

**RCL Amplification and Detection**

***Positive Standard***
***FeLV B/C***
**Efficient amplification**
**  ****in 293 & lymphoid cells**
** ****Regulatory standard**
** ****No LentiVector **
**  ****interference**

***HEK293***
**Tranduced by**
**   ****wide range of**
**  ****Vectors and**
**  ****pseudotypes**
***Lymphoid (Raji, H9, Sup1)***** **
** ****Potential in-patient**
**  ****target for RCLs**

**Notes:**
An overview of our RCL testing process is shown.

The PERT and PCR tests are validated with FeLV to detect a single RCR in 100ml  using various spiked mixtures to guard against vector interference. The LentiVector test article is amplified. Current MLV production protocols specify 14 days for amplification and we propose that we follow the MLV model.

Two human cell types are used for amplification. We are currently evaluating different lymphoid lines. These will give an indication of any genetic interaction that might occur in the patient where PBMCs are a potential target for replication. 

## Minimising the Generation 

** ****Split the vector production system into at least three components**
** ****Preferably use a stable producer cell line**
** ****Eliminate all non-essential coding and *****cis*****-active sequences**
** ****Minimise the potential for homologous recombination**
** ****Reduce packaging of vector helper components**
** ****Use sensitive, calibrated assays for RCLs after amplification**
**  ****in human cells**

**Minimising the Generation **
**and Impact of RCL**

**Oxford BioMedica proposes that this is a specification for discussion**

**Notes:**
We have made considerable progress in minimising the potential impact and generation of RCLS in an EIAV based vector production system. We have devised a quantitative biochemical assay for screening for RCLs and a second line biochemical assay for resolving any ambiguities. This assay strategy needs to be debated and may require modification and additions. The current strategy is now being validated at scale with benchmark producer lines.

## LentiVectors : 

**Key Vector-Specific Safety Issues**

**Replication Competent Lentiviruses in the product**
***Minimise the generation and impact of RCLs***
**Mobilisation of the transfer vector in target cells**

***Minimise the inappropriate dissemination of the transgene***

**Notes:**
Now we turn to the second vector-specific safety issue. This is the potential for transfer vector mobilisation.

## Minimising Mobilisation

**Use a transfer vector that is poorly mobilised**
**Reduce the level of mobilisable RNA in the target cell **

**Notes:**
Depending upon the therapeutic gene it may be undesirable for it to be transferred to other cells or indeed outside of the patient.  For example an anti-apoptotic gene or growth factor is classified as having a high damage potential and needs to be contained. In this case the transfer vector mobilisation potential will need careful scrutiny. In contrast a pro-drug activating enzyme that is non-functional in the absence of the pro-drug is classified as having a low damage risk and the requirement for containment is consequently lower. In  this case mobilisation may be less of an issue.
We can envisage two general strategies to minimise the potential for mobilisation of the transfer vector.

## Minimising Mobilisation

**HIV-1 is the agent for mobilisation that is of most concern**

**Use a transfer vector that is poorly mobilised**

**Notes:**

It is not possible to predict all scenarios for mobilisation. We have therefore chosen to consider mobilisation by HIV-1 as the most extreme case and one that is likely to be of most concern. We have compared this with mobilisation by MLV as an example of a generic retrovirus. We consider that other scenarios such as mobilisation by endogenous elements are unlikely to occur with greater efficiency than our chosen test systems.

## LentiVectors: Cross - Packaging Assays

**LentiVectors: Cross - Packaging Assays**

**HEK293T**

**HIV**

**MLV**

**Gag-Pol**

**GFP**

**Biological titre on D17 cells**
**by FACS for GFP after 5d**
**And after 2 serial passages**

**= 1**

**= 1**

**= 1**

**1/4**

**1/4**

**Test pairwise combinations for packaging and transduction**

**Virion associated**
**GFP RNA(TaqMan)**

**Env**

**Notes:**
We have devised a simple assay to assess the mobilisation potential of EIAV vectors.
HEK293 cells are transfected with VSV-G and with HIV, MLV or EIAV transfer vectors expressing GFP together with HIV, EIAV or MLV Gag-Pol plasmids in all pairwise combinations (9). The amount of GFP RNA in particles is measured by TaqMan. The transducing capability of that GFP RNA is measured on sensitive D17 cells by FACs on initially transduced cells and after passage to exclude pseudo-transduction. 

## LentiVector and Retroviral Vector

** ****Cross Transmission: Initial Titres**

**EIAV**

**HIV**

**MLV**

- Titre (TU per ml)

**Gag-Pol**

**EIAV genome**

**MLV genome**

**HIV genome**

** ****Cross mobilisation between EIAV and HIV occurs at less than 1000 fold **
**  ****the efficiency of homologous mobilisation**
** ****EIAV and MLV are similarly poorly mobilised by HIV**
** ****NB: MLV is already  in the clinic in HIV+ve patients**

**Notes:**
Due to time constraints only the data for cross transmission are shown but these mirror cross packaging.  EIAV is mobilised by HIV and MLV at 1000 fold lower efficiency than the cognate pairs. Interestingly EIAV is not significantly different from MLV in mobilisation by HIV. This suggests that no special precautions are needed with respect to HIV status of patients when EIAV is translated to the clinic as none are currently applied to MLV.

EIAV does not mobilise MLV to a significant extent and acts on HIV with 1000 fold lower efficiency than the cognate pairs.

It is interesting to note that there is a background mobilisation in all combinations and this is to a degree consistent with the background packaging of cellular RNA that we found in the packaging assays. There are however some apparent preferential interactions as the HIV RNA is packaged more promiscuously and the HIV Gag-Pol packages somewhat more promiscuously. These promiscuous events still represent a 1000 fold lower efficiency than the cognate pair.  It is difficult to see how to reduce this background further.

## Minimising Mobilisation

**Reduce the level of mobilisable RNA  in the target cell**

** ****Achieved by modifying the transfer vector e.g. SIN vectors**
** ****Should not be required for LentiVectors that are already poorly mobilised**

**Notes:**
If mobilisation by HIV-1 is below an acceptable, defined and quantifiable limit then further vector manipulation should not be necessary. We propose that the limit is broadly defined by the properties of MLV. If a LentiVector displays a similar order of  mobilisation by HIV-1 as MLV then, we would argue, there is no absolute need for further modification. This would bring LentiVectors into line with retroviral vectors where it has been possible to add modifications such as self inactivation (SIN) for many years but this is not a regulatory requirement for clinical evaluation to proceed. 

Despite the poor mobilisation of EIAV by HIV-1 we have still explored the possibility for further vector modification. This may become appropriate for particular gene and protocol configurations.

## Self Inactivating (SIN) EIAV  Vectors

**Self Inactivating (SIN) EIAV  Vectors**

**pONY8.1 SIN Vector: Configuration in the transduced target cell**

** ****Very low level expression **
**of genomic RNA **

** ****Residual viral genome is 900nt**
** ****Deletes the LTR: no evidence that this deletion is beneficial *****per se***** and it reduces**
** ****vector options for second transcription units.**
** ****SIN transfer vectors cannot be introduced into packaging cells by transduction unless a**
**  ****conditional SIN is used**

**mRNA in target cell**

**CMVp**

*****

**SIN LTR**

**LacZ**

**Notes:**
The SIN strategy is well known for MLV vectors and has more recently been adapted for HIV-1 based vectors. The strategy exploits the reverse transcription pathway. Basically once the transfer vector has integrated into the target cell the sequences that were originally present at the 3’ end have been copied during provirus formation to the 5’end. All efficient promoters that were used to drive the production of the transfer vector RNA in the production cell are therefore lost unless they were also present in the 3’ LTR. Likewise any deletion of the 3’ LTR or any addition of a new promoter will be transferred to the 5’ end in the transduced cell. In this way all promoter activity can be abolished as in a SIN configuration or a new promoter e.g. a cell-specific or regulated promoter can be created.
The EIAV LTR is a natural SIN in human cells as it requires Tat and EIAV Tat cannot interact with human cyclin. We have however deleted all enhancers and the TATA box in the LTR to make a classical SIN vector. 
This has considerable operational disadvantages in that it reduces the further design flexibility of the vector by preventing the use of regulated or  tissue specific promoters at this site and  it forces the use of transfection or more complicated conditional SIN vectors to make the producer cell line which may impact on efficiency.

## Performance of EIAV LTR and SIN Vectors

**RNA levels in the target cell by at least 3 logs. **

**Performance of EIAV LTR and SIN Vectors**

**Notes:**
We have examined the levels of potentially packageable RNA that is produced from  integrated SIN, LTR or CMV vectors using TaqMan and a  probe. These RNAs could initiate within the vector, externally depending upon the integration site and be partial transcripts. The tranducing potential of these RNAs is measured by obtaining a LacZ transducing titre on sensitive D17 cells.

There is no difference between the LTR and SIN. Both produce 1000 fold less packageable RNA and both reduce the transducing titre in this experiment by about 5 logs.
It is therefore possible to reduce the potential for mobilisation of an EIAV vector to below that of MLV. It is not clear why this should be required and it would impose limitations on vector development.  

We propose that the requirement for SIN vectors is assessed on a case by case basis that balances disadvantages and benefit. 

## Mobilisation of HIV Vectors by HIV

**A special case for the treatment of AIDS?**

**Turns virus escape into an attribute**

**Notes:**
Gene therapy strategies are being developed to combat AIDS. The aim is to reduce viral load. Ideally the therapy would act at a stage before viral integration. However if this is not done then there is the risk that HIV will escape the therapy. It may be possible to ensure that escape virus carries within it the seeds of its own destruction.

## ImmStat: A Mobilisable Therapeutic 

**Rz Vector for the treatment of AIDS **

** ****pSYNGP allows Rz directed against the conserved *****gag-pol***
** ****No immune responses anticipated against therapeutic RNA**
** ****Mobilisation amplifies the therapy if virus escapes the first wave of Rzs**

**Tat inducible**
** ****genomic and**
** ****therapeutic RNA**

**ImmStat Vector Particle**

**Constitutive**
** ****therapeutic RNA**

****

**Anti-HIV and chemokine **
**receptor Ribozymes**

**CMV**

**R-U5**

****

**Multi-Rz**

**CMVp**

**LTR**

**Notes:**
ImmStat is configured as an HIV vector for one major reason and that is that the transfer vector can be mobilised. Tat regulation can be achieved in other vectors, retroviral vectors can transduce CD34 cells and other lentiviral vectors might be able transduce repopulating stem cells, although at present HIV is the best characterised for this application. If mobilisation is not seen as an advantage then the choice of HIV vectors to treat HIV may be less compelling.

## HIV-1 Production from Infected ImmStat

**HIV-1 Production from Infected ImmStat**
**Positive and Negative cells**

- 0

- 5000

- 10000

- 15000

- 20000

- 25000

- 30000

- 0

- 2

- 4

- 6

- 8

- 10

- 12

- 14

- 16

- 18

- 20

- 22

- 24

- Days

- RT Units

**HIVGFP**

**HIVGFP- ImmStat**

**ImmStat delays HIV replication **

**? Genetic variant ?**

**Notes:**
HIV always escapes gene therapy in vitro. This is assumed to be due to the non-physiological conditions, i.e. titre, cell proximity , lack of dilution etc.  It would however be prudent to confirm the genetic identity of escapes to ensure that there is no selection by the gene therapy per se.

Various issues have been raised concerning the VIRxSys proposal. We believe that it is possible to design an HIV based vector for the treatment of AIDS. However attention should be paid to minimise the potential for RCLs not just in production but to avoid novel RCLs in the co-infected patient’s cells. In particular the genetic nature of any escape virus could be evaluated in a model system. 

In addition the potential for toxicity of the therapeutic cargo beyond the intended target cell could be evaluated if mobilisation is a feature of the vector system. If mobilisation by the patient’s virus is not a requisite part of the protocol then the benefits of using an HIV based vector could be demonstrated. 

## LentiVectors : 

**Key Vector-Specific Safety Issues**

**Replication Competent Lentiviruses in the product**
***Minimise the generation and impact of RCLs***
**Mobilisation of the transfer vector in target cells**
***Minimise the inappropriate dissemination of the transgene***

**Notes:**
After that brief digression into HIV I would like to summarise our findings with the EIAV system. We believe that we have made considerable progress in addressing both of the key vector-specific safety issues, particularly with EIAV vectors.

## A LentiVector System With a 

**Good Safety Profile**

***pONY8.1 Z or G***

***pTOG***

***pESYNGP***

**pA**

**  ****No pathogenic proteins**
**  ****Very poorly mobilised by HIV (sim. MLV)**
**  ****No potential for homologous recombination**
**  ****RCLs extremely unlikely**

****

**WPRE**

**cPPT**

**Lac Z**

**LTR**

**CMVp**

**Notes:**
We have described strategies to generate LentiVectors with a high safety profile. These strategies could be applied to the development of vectors based on any lentivirus. We have made some progress in applying these strategies to HIV vectors and believe that such vectors will have role to play in developing treatments for AIDS.

## LentiVector Specification: A Proposal

**LentiVector Specification: A Proposal**

***  ******A LentiVector system must show efficacy in a relevant animal model***
***  ******It must be capable of scale production at GMP***
***  ******It should be configured with the following points in mind***

**1.****	****Eliminate non-essential proteins and sequences.**
**2.****	****Ensure extremely low, preferably zero, homologous recombination potential between vector components.**
**Show significant differential between self mobilisation and mobilisation with HIV or, use a vector that after integration generates a significant reduction in mobilisable RNA.  Use MLV as a benchmark for mobilisation.**
**Use a minimum of three split components in a stable packaging cell line**
**	****or in a very low-homologous-recombination transient system.**
**Use sensitive, calibrated assays for RCLs (follow MLV guide) with an amplification step in an appropriate human cell line.**
**6.****	****Test for no significant acute toxicity in the animal efficacy model.**

**Notes:**
We believe that we are coming close to describing a basic generic specification for a LentiVector. The refinement of this specification is an ongoing process and this meeting is an important step in the process. The UK Gene Therapy Advisory Council GTAC  are setting up a working group to review the issues surrounding the translation of these powerful new vectors to the clinic and the first meeting will be early in December this year. We hope that a set of  guidelines for the safe design and testing of LentiVectors will soon emerge and that we can proceed to test these vectors in the clinic.

## Supporting References

**on human immunodeficiency virus type 1. J. Virol. **_**72**_**, 811**
**K.A. Mitrophanous et al (1999) Stable gene transfer to the nervous **
**system using a non-primate lentiviral vector**
**E. Kotsopoulou et al (2000) A Rev-independent human immunodeficiency**
**virus type 1 –based vector that exploits a codon-optimised *****gag-pol***** gene.**
**J. Virol. **_**74**_**, 4839**
**N.D. Mazarakis et al (2001) Rabies virus pseudotyping of lentiviral vectors**
**enables retrograde axonal transport and acccess to the nervous system after **
**peripheral delivery. Human Molecular Genetics, **_**10**_**, 2109.**
**E.Martin-Rendon, et al (2001) Lentiviral vectors for the treatment of **
**Neurodegenerative diseases. Curr. Opinion Molecular Therapeutics, **_**3**_**, 476.**

**Supporting References**

## The LentiVector  Team

**Virology**
**Kyri Mitrophanous**
**Jonathan Rohll**
**Fiona Ellard**
**Fraser Wilkes**
**Anna Olsen**
**Pippa Radcliffe**
**James Miskin**
**Danny Chipchase**
**Margaret Esapa**

**Neurobiology**
**Nick Mazarakis**
**Enca Martin-Rendon**
**Mimoun Azzouz**
**Karen Vincent**
**Rob Barber**

**Production**
**Andrew Slade**
** ****Wendy Blakemore**
**Jay Brown**
**Linda Dove**

**Collaborators**
**John Olsen **
**Karen O Malley **
**James Uney**
**LGC**
**NIBSC**
**Q-One Biotech**

**BioMedica Inc**
**Doug Jolly**
**Holger Roehl**

**Consultants**
**Jonathon Weber**
**Mark Saunders**
**Robin Weiss**
**Mary Collins**
**Patrick Aebischer**
**Krys Bankiewicz**

**Biological **
**Systems**
**Stuart Naylor**
**Katie Binley**
**On Kan**
**Leigh Griffiths**
**Emma Carter**
**Zoe Askham**